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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2385
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3544-3549
Copyright © 2005 by The Endocrine Society

Preproorexin and Orexin Receptors Are Expressed in Cortisol-Secreting Adrenocortical Adenomas, and Orexins Stimulate in Vitro Cortisol Secretion and Growth of Tumor Cells

R. Spinazzi1, M. Rucinski1, G. Neri, L. K. Malendowicz and G. G. Nussdorfer

Department of Human Anatomy and Physiology (R.S., G.N., G.G.N.), Section of Anatomy, University of Padua, I-35121 Padua, Italy; and Department of Histology and Embryology (M.R., L.K.M.), Poznan School of Medicine, PL-60781 Poznan, Poland

Address all correspondence and requests for reprints to: Professor G. G. Nussdorfer, Department of Human Anatomy and Physiology, Section of Anatomy, University of Padova, Via Gabelli 65, I-35121 Padova, Italy. E-mail: gastone.nusdorfer{at}unipd.it.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Orexins A and B are hypothalamic peptides that originate from the proteolytic cleavage of preproorexin and act through two subtypes of receptors, named OX1-R and OX2-R. OX1-R almost exclusively binds orexin-A, whereas OX2-R is nonselective for both orexins. We previously found that orexin-A, via the OX1-R, stimulates cortisol secretion from dispersed human adrenocortical cells. In this study, we demonstrate that six of eight cortisol-secreting adenomas expressed preproorexin mRNA, and seven of 10 adenomas contained measurable amounts of orexin-A but not orexin-B. Normal adrenal cortexes neither expressed preproorexin nor contained orexins. All adenomas expressed OX1-R and OX2-R mRNAs, and real-time PCR showed that the expression of both receptors was up-regulated in adenomas, compared with normal adrenal cortex. Orexin-A concentration-dependently raised basal cortisol secretion from freshly dispersed normal and adenomatous cells, minimal and maximal effective concentrations being 10–10 and 10–8 M, and the peptide efficacy (percent increase elicited by 10–8 M orexin-A) was significantly higher in adenomas than in the normal adrenal cortex. Orexin-B was ineffective, thereby indicating that orexin secretagogue action is mediated by the OX1-R. In contrast, both orexins (10–8 M) raised the proliferative activity of cultured normal and adenomatous cells, suggesting that this effect is mediated by OX2-R or both receptor subtypes. Collectively, our findings allow us to conclude that the orexin system is overexpressed in cortisol-secreting adenomas and suggest that orexin-A may act as an autocrine-paracrine regulator of the secretory activity and growth of some of these adrenal tumors.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
OREXINS A AND B ARE hypothalamic peptides involved in the central regulation of feeding and sleep (1, 2, 3). They originate from the posttranslational proteolytic processing of preproorexin, and bind two subtypes of G protein-coupled receptors, named OX1-R and OX2-R (4, 5, 6).

Evidence has been provided that orexins stimulate glucocorticoid secretion from dispersed rat and human or cultured pig adrenocortical cells (7, 8, 9, 10), and, accordingly, OX1-R and OX2-R expression has been detected in adrenocortical cells (9, 10, 11, 12, 13, 14). Frequently, peptides modulating the adrenocortical function are locally synthesized in the gland, thereby acting in an autocrine-paracrine manner (for review, see Ref. 15). Consistent with this contention, a weak preproorexin expression has been demonstrated in human adult and fetal adrenals (12, 14, 16).

Investigations on the orexin and orexin receptor expression and function in adrenocortical tumors are very rare: the only presently available finding is that both cortisol- and aldosterone-secreting adenomas are provided with OX1-Rs (14). It seemed therefore worthwhile to study whether adrenocortical adenomas express orexin and its receptors and possess secretory and growth in vitro responses to orexins.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Reagents

Orexin-A and orexin-B were purchased from Phoenix Pharmaceuticals (Belmont, CA), and goat polyclonal antibodies against OX1-Rs and OX2-Rs from Santa Cruz Biotechnology (Santa Cruz, CA). Medium 199 was provided by Difco (Detroit, MI). SQ-22536, U-73122, H-89, and calphostin-C were obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Human serum albumin, fetal calf serum, DMEM, PBS, 5'-bromo-2'-deoxyuridine (BrdU), and all other chemicals and laboratory reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO).

Patients

Seven patients with unilateral cortisol-secreting adrenal adenomas who had produced Cushing’s syndrome were recruited. They displayed high basal levels of plasma and urinary 17-hydroxysteroids and 17-ketosteroids and low levels (<8 pg/ml) of plasma ACTH. The patients underwent surgery, and resected tumors were histologically identified as lipid-rich cell, benign adrenocortical adenomas (adenomas 1–5 and 9 and 10). Fragments of adrenal tails, which do not contain medullary chromaffin cell tissue (17), were obtained from six adult patients undergoing nephrectomy/adrenalectomy for kidney cancer (adrenals 1–4 and 9 and 10). Each patient gave written informed consent, and the study protocol was approved by the local ethics committee for human studies. Adrenal specimens were collected immediately after excision in the operating room, placed in Krebs-Ringer bicarbonate buffer with 0.2% glucose at 4 C, and immediately carried to our laboratory. Each specimen was partly frozen at –80 C and partly immediately used for functional experiments.

RT-PCR

Total RNA was extracted from frozen adrenal specimens (adenomas 1–5 and adrenal tails 1–4) as well as stored frozen fragments of the three cortisol-secreting adenomas and four adrenal tails (specimens 6–8 and 5–8, respectively) used in previous studies (18, 19) and reverse transcribed to cDNA (20). PCR was performed as detailed earlier (21), using the primers for human preproorexin, OX1-R and OX2-R published by Sakurai et al. (4) and Nakabayashi et al. (16) (Table 1Go). In a Delfi 100 thermal cycler (MJ Research Inc., Waterston, MA), we used the following PCR programs: 1) preproorexin, 37 cycles of 96 C for 30 sec, 60 C for 60 sec, and 72 C for 120 sec; and 2) OX1-R and OX2-R, 38 cycles of 95 C for 60 sec, 59 C for 60 sec, and 72 C for 60 sec. An additional extension step at 72 C for 7 min was then carried out. As positive control, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected. To rule out the possibility of amplifying genomic DNA, one PCR was carried out without prior reverse transcription (RT) of the RNA. Detection of the PCR amplification products was performed by size fractionation on 2% agarose gel electrophoresis. Identification of amplicons was subsequently confirmed by sequencing (Alf sequencer; Pharmacia Biotech, Freiburg, Germany).


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  		TABLE 1. RT-PCR primers and PCR products

 
Real-time PCR

The relative expression of OX1-R and OX2-R mRNAs in cortisol-secreting adenomas (specimens 2–5) and adrenal tails (specimens 1–4) was assayed in an I-Cycler iQ detection system (Bio-Rad Laboratories, Milan, Italy), using the primers shown in Table 1Go, and the following protocol: denaturation step (95 C for 3 min), 38 cycles of two steps of amplification (95 C for 15 sec and annealing for 30 sec), and melting curve (60–90 C with a heating rate of 0.5 C/10 sec). During the exponential phase, the fluorescence signal threshold was calculated, and the fraction number of PCR cycles required to reach the threshold (cycle threshold) was determined. Cycle threshold values decreased linearly with increasing input target quantity and were used to calculate the relative mRNA expression. The specificity of amplification was tested at the end of each run by melting-curve analysis, using the I-Cycler iQ software 3.0. All samples were amplified in duplicate, and GAPDH was used as reference to normalize data.

Western blotting

Frozen fragments of cortisol-secreting adenomas (specimens 1–5, 9, and 10) and adrenal tails (specimens 1–4, 9, and 10) were lysed in ice-cold radioimmunoprecipitation assay buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 1% sodium dodecyl sulfate). Phenylmethylsulfonylfluoride, sodium orthovanadate, and apoprotein were added to inhibit proteinase activity. One hundred micrograms of proteins from each lysate were used for SDS-PAGE. The proteins were first reduced with sample buffer [4% sodium dodecyl sulfate, 20% glycerol, 0.5% ß-mercaptoethanol, and 125 mM Tris (pH 6.8)] and then boiled for 5 min before loading on 12.5% polyacrylamide gel. Cruz Marker MW standard (Santa Cruz Biotechnology) was used to assess the size of proteins of interest. Samples were resolved in a standard gel apparatus and then transferred to nitrocellulose membranes (Sigma-Aldrich). Membranes were blocked for 60 min in Blotto A with 0.05% Tween 20 and incubated for 120 min with primary goat polyclonal anti-OX1-R (C19) and anti-OX2-R (N20) antibodies, 1:1000 diluted in Blotto A. After washing in Tris-buffered saline buffer, membranes were incubated for 60 min with Cruz Marker-compatible peroxidase-conjugated secondary antibodies (1:2000 dilution). Immunoreactive bands were detected using the chemiluminescent luminol reagent (Santa Cruz Biotechnology) and exposed to autoradiography film (Eastman-Kodak, Rochester, NY).

Orexin concentration assay

Fragments of cortisol-secreting adenomas (specimens 1–10) and adrenal tails (specimens 1–10) (about 200 mg of tissue) were extracted as described by Arihara et al. (22), and orexin-A and orexin-B concentrations were measured, without previous purification, using commercial RIA kits provided by Phoenix Pharmaceuticals. The following were determined for the orexins: orexin-A RIA kit: sensitivity, 5 fmol/tube; cross-reactivity, orexin-A (human, mouse, and rat) 100%, and orexin-B and other peptides 0%; intra- and interassay coefficients of variation, 7.5 and 8.7%, respectively; orexin-B RIA kit: sensitivity, 6 fmol/tube; cross-reactivity, orexin-B (human, mouse, and rat) 100%, and orexin-A and other peptides 0%; intra- and interassay coefficients of variation 8.0 and 9.2%, respectively. As positive control, orexin concentrations were measured in the hypothalamus of six rats.

In vitro secretion experiments

Dispersed human adrenocortical and tumor cells were obtained from fresh cortisol-secreting adenomas (specimens 1–5) and adrenal tails (specimens 1–4) by sequential enzymatic digestion and mechanical disaggregation (18). Dispersed cells were put in medium 199 and Krebs-Ringer bicarbonate buffer with 2% glucose, containing 5 mg/ml human serum albumin, and incubated (5 x 104 cells/ml) with increasing concentrations (from 10–12 to 10–6 M) of orexin-A or orexin-B. Dispersed cell preparations obtained from cortisol-secreting adenomas (specimens 4, 5, 9, and 10) and adrenal tails (specimens 3, 4, 9, and 10) were incubated with 10–8 M orexin-A in the presence of 10–4 M SQ-22536 [that inhibits agonist-induced cAMP production (IC50, 1 µM)] or 10–5 M U-73122 [that inhibits agonist-induced phospholipase C activation (IC50, 1.0–2.1 µM)], H-89 [that inhibits protein kinase (PK)A (IC50, 0.05 µM)], or calphostin-C [that inhibits PKC (IC50, 0.05 µM)]. The incubation was carried out in a shaking bath at 37 C for 60 min in an atmosphere of 95% air-5% CO2. At the end of the experiments, the incubation tubes were centrifuged at 4 C at 100 x g for 10 min, and supernatants were stored at –80 C. Cortisol was extracted from the incubation medium and purified by HPLC (23). Its concentration was measured by RIA with a commercial kit provided by IRE-Sorin (Vercelli, Italy). Sensitivity was 90 pmol/liter; intra- and interassay coefficients of variation were 6.5 and 7.9%, respectively.

In vitro growth experiments

Dispersed cells obtained from cortisol-secreting adenomas (specimens 5, 9, and 10) and adrenal tails (specimens 4, 9, and 10) were suspended in DMEM, containing 20% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin and plated in 35-mm tissue culture dishes. Cells were seeded at a density of 104 cells/dish, and cultured at 37 C in a humidified atmosphere of 95% air-5% CO2 for 24 h (24). Three nonconfluent cultures obtained from each specimens, were incubated for a further 24 h at 37 C in fresh DMEM with 10–8 M orexin-A or orexin-B. Other cultures dishes were incubated without orexins (baseline values). During the last 12 h of incubation, BrdU was added to the culture medium at a final concentration of 10 µM (24). Cultures were fixed in 4% paraformaldehyde for 30 min. BrdU-positive (S-phase) cells were detected using a cell proliferation kit (Amersham Pharmacia, Aylesbury, UK).

Statistics

Data were expressed as the mean ± SEM or SD of five, four, or three independent experiments, each experiment being performed with a cell suspension or cultures obtained by a single adrenal gland or adenoma. Statistical analysis was carried out by ANOVA, followed by Duncan’s multiple range test.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
RT-PCR showed the expression of preproorexin mRNA in six of the eight adenomas but not in the normal adrenal cortexes. GAPDH mRNA was detected in all specimens (Fig. 1Go). RIA demonstrated measurable levels of orexin-A in seven (means ± SD, 12.2 ± 4.1) of the 10 adenomas (Table 2Go), whereas the peptide concentration in all normal adrenocortical-tissue samples was below the limit of sensitivity of our assay. Orexin-B concentration was not measurable in either normal or adenomatous specimens. As expected, high concentrations of both orexins were present in the rat hypothalamus (Table 2Go). OX1-R and OX2-R mRNAs were demonstrated by conventional PCR in both adenomas and adrenocortical tissue (Fig. 2Go). Real-time PCR showed that the expression of both OX1-R and OX2-R mRNAs was markedly higher in adenomas than the normal adrenal cortex (Fig. 3Go). Western blotting demonstrated the presence of OX1-R and OX2-R proteins in the lysates of both tissues (single band proteins of about 50 and 40 kDa), and the specificity of the reaction was confirmed by the virtually lack of immunodetection when antibodies were preabsorbed with their respective blocking peptides (Fig. 4Go).



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  		FIG. 1. Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human preproorexin-specific primers from RNA of normal adrenal cortexes (specimens 1–8, upper panel) and cortisol-secreting adrenocortical adenomas (specimens 1–8, lower panel). Lane 1 was loaded with 200 ng of a size marker (Marker VIII; Roche, Mannheim, Germany). The amplified fragments were of the expected sizes, which were 480 bp for preproorexin and 100 bp for GAPDH. No amplification without prior RT of RNA is shown as a negative control.

 

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  		TABLE 2. Orexin-A and orexin-B concentrations (fmol/mg) in cortisol-secreting adrenocortical adenomas

 


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  		FIG. 2. Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human OX1-R- and OX2-R-specific primers from RNA of normal adrenal cortexes (specimens 1–8, upper panel) and cortisol-secreting adrenocortical adenomas (specimens 1–8, lower panel). Lane 1 was loaded with 200 ng of a size marker (Marker VIII, Roche). The amplified fragments were of the expected sizes, which were 189 bp for OX1-R, 201 bp for OX2-R, and 585 bp for GAPDH. No amplification without prior RT of RNA is shown as a negative control.

 


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  		FIG. 3. Real-time PCR of OX1-R and OX2-R mRNAs in adrenal cortex (specimens 1–4) and cortisol-secreting adenomas (specimens 2–5). Bars, means ± SEM (n = 4). *, P < 0.05; and **, P < 0.01 vs. adrenal cortex.

 


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  		FIG. 4. Western blot analysis of OX1-R and OX2-R in exemplary protein extracts from adrenal cortex (a) and cortisol-secreting adenoma (b). OX1-R and OX2-R proteins are recognized as single bands of about 50 and 40 kDa, respectively. The specificity of detection is demonstrated by the absence of immunodetection when antibodies were preabsorbed with their respective blocking peptides (lanes c and d for adrenal cortex and adenomas, respectively). Molecular mass standards (kilodaltons) are shown in lane 1.

 
Orexin-A, but not orexin-B, concentration-dependently increased basal cortisol secretion from freshly dispersed normal and adenomatous cells, the minimal and maximal effective concentrations being 10–10 and 10–8 M (Fig. 5Go). Orexin-A efficacy (percent increase elicited by the maximal effective concentration) was significantly higher in adenomas than the normal adrenal cortex [435 ± 52 vs. 237 ± 39 (P < 0.01)], whereas the potency (half-maximal concentration) did not differ [4.72 ± 0.45 vs. 5.41 ± 1.1 x 10–10 M (P > 0.2)]. The cortisol secretagogue effect of 10–8 M orexin-A was abolished by either the adenylate cyclase inhibitor SQ-22536 (25) or the PKA inhibitor H-89 (26) at concentrations (10–4 and 10–5 M, respectively) that were previously found to suppress the cAMP-dependent cascade (19, 24). In contrast, it was unaffected by both the inhibitor U-73122 (27) and the PKC inhibitor calphostin-C (28) (Fig. 6Go).



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  		FIG. 5. Effects of orexin-A (upper panel) and orexin-B (lower panel) on basal cortisol production from dispersed human adrenocortical (specimens 1–4) (open circles) and cortisol-secreting adenoma cells (specimens 1–5) (solid circles). Points are the mean ± SEM of four or five separate experiments. *, P < 0.05; and **, P < 0.01 vs. the respective baseline value (B).

 


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  		FIG. 6. Effects of SQ-22536 (10–4 M), H-89 (10–5 M), U-73122 (10–5 M), and calphostin-C (10–5 M) on basal and orexin-A (10–8 M)-stimulated cortisol secretion from dispersed human adrenocortical (specimens 3, 4, 9, and 10) (upper panel) and cortisol-secreting adenoma cells (specimens 4, 5, 9, and 10) (lower panel). Bars, Mean ± SEM of four separate experiments. **, P < 0.01 vs. the respective baseline value; a, P < 0.01 vs. the respective control value.

 
Both orexin-A and orexin-B (10–8 M) raised the proliferative activity of cultured human normal and adenomatous cells. Apparently orexin-B was more effective than orexin-A, and the proliferogenic effect of both orexins was more intense in cultured adenomatous than normal adrenocortical cells (Fig. 7Go).



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  		FIG. 7. Effects of orexin-A and orexin-B (10–8 M) on the proliferative activity of cultured human adrenocortical (specimens 4, 9, and 10) and cortisol-secreting adenomas (specimens 5, 9, and 10). Bars, Means ± SD of three separate experiments. *, P < 0.05; and **, P < 0.02 vs. the respective control value.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Our study provides novel evidence that some cortisol-secreting adrenocortical adenomas express preproorexin mRNA and contain measurable amounts of orexin-A immunoreactive protein. In contrast, orexin expression, either as mRNA or protein, was not detected in normal human adrenal cortexes. Sizable levels of orexin-A protein were not previously detected in six cortisol-secreting adrenocortical adenomas (22), whereas a low expression of preproorexin and orexin-A proteins and preproorexin mRNA were demonstrated by Western blotting and PCR in human adrenals (12, 14, 16). We were unable to explain these discrepancies. However, we stress that they could be ascribed to the variability of adrenocortical adenomas, the different sensitivity of the RIAs used, and the fact that previous findings were obtained using entire adrenal glands, i.e. containing medullary chromaffin tissue that is able to synthesize a lot of regulatory peptides (15). Another important issue addressed by our investigation is that cortisol-secreting adenomas, like human adrenal cortex (9), express both OX1-R and OX2-R as mRNA and protein and not only OX1-Rs as reported previously (14). Moreover, real-time PCR clearly demonstrated that the expression of both orexin-receptor subtypes is up-regulated in adenomas.

Orexin-A concentration-dependently stimulates basal cortisol secretion from both normal and adenomatous freshly dispersed adrenocortical cells, whereas orexin-B is ineffective. Compelling evidence indicates that OX1-Rs prevalently, if not exclusively, bind orexin-A, whereas OX2-Rs are not selective for both orexins (4, 5, 6). Hence, the lack of any secretory effect of orexin-B, which does not activate OX1-R, confirms the contention that the glucocorticoid secretagogue action of orexins in rats and humans are almost exclusively mediated by the OX1-R subtype (8, 9). Of great interest is that the efficacy, but not the potency, of orexin-A is significantly higher in dispersed adenomatous than normal adrenocortical cells, which accords well with the presently demonstrated up-regulation of OX1-R expression in the tumor tissue. According to some findings in the normal adrenal cortex (8), our results strongly suggest that also in adenomas the OX1-R-mediated cortisol secretagogue action of orexin-A exclusively involves the activation of the adenylate cyclase-PKA cascade. In fact, both the adenylate cyclase inhibitor SQ-22536 and the PKA inhibitor H-89 suppressed the secretory response to orexin-A, whereas the phospholipase C inhibitor U-73122 and the PKC inhibitor calphostin-C were ineffective. Neither SQ-22536 nor H-89 per se evoked significant changes in cortisol secretion, thereby ruling out the possibility that their effect was due to a nonspecific toxic lesion of the steroidogenic machinery.

Our PCR findings show that not only OX1-Rs but also OX2-Rs are expressed in normal and adenomatous adrenal tissue. Previous studies demonstrated that OX2-Rs are involved in the mediation of the orexin-modulating action on the secretory activity of rat PC12 cells (29) and human pheochromocytomas (30), but this does not seem to be case in adrenocortical cells, in which only OX1-Rs are involved in the secretagogue effect of orexins. Our findings, although obtained in only three adenomas and three normal adrenal cortexes, suggest that OX2-Rs, alone or in cooperation with OX1-Rs, are involved in a growth-promoting effect of orexins. In fact, both orexins were found to enhance the proliferative activity of cultured normal adrenocortical and adenomatous cells, and orexin-B exclusively binds OX2-R, whereas orexin-A binds both receptor subtypes (8, 9). This last contention may also explain why, at an equimolar concentration, orexin-B was more effective than orexin-A in stimulating cell growth. Moreover, real-time PCR findings, indicating that not only OX1-Rs but also OX2-R expression are up-regulated in cortisol-secreting tumors, are in good agreement with the observation that the proliferogenic action of orexins was more intense in tumor than normal adrenocortical cell cultures.

The physiological relevance of our findings remains to be ascertained. Under basal conditions, the role of orexins as modulators of adrenocortical glucocorticoid secretion is very doubtful, inasmuch as the minimum effective concentration of orexin-A eliciting an in vitro secretory response (10–10 M) is about 2 orders of magnitude higher than its level in the blood of normal human volunteers (31). In contrast, orexins may be relevant in the functional regulation of cortisol-secreting adenomas, which not only highly express orexin receptors but also express preproorexin gene and contain sizable amounts of orexin-A. According to Nussdorfer (15), it may be calculated that the adenoma content of orexin-A, as measured by RIA in the present investigation, may give rise to a local concentration ranging between 10–8 and 5 x 10–8 M. This figure is surely compatible with an autocrine-paracrine mechanism of action of orexin-A in the control of secretion and growth of some cortisol-secreting adrenal tumors.


   Acknowledgments
 
We thank Ms. A. Coi for her secretarial support and invaluable help in the provision of bibliographic items.


   Footnotes
 
First Published Online March 29, 2005

1 R.S. and M.R. should be considered first coauthors. Back

Abbreviations: BrdU, 5'-Bromo-2'-deoxyuridine, GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OX1-R, orexin receptor type 1; OX2-R, orexin receptor type 2; PK, protein kinase; RT, reverse transcription.

Received December 8, 2004.

Accepted March 22, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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