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Orexins stimulate glucocorticoid secretion from cultured rat and human adrenocortical cells, exclusively acting via the OX1 receptor
Journal of Steroid Biochemistry & Molecular Biology 96 (2005) 423–429
Orexins stimulate glucocorticoid secretion from cultured rat and human adrenocortical cells, exclusively acting via the OX1 receptor Agnieszka Ziolkowska a , Raffaella Spinazzi b , Giovanna Albertin b , Magdalena Nowak a , Ludwik K. Malendowicz a , Cinzia Tortorella b , Gastone G. Nussdorfer b,∗ a
Department of Histology and Embryology, Poznan School of Medicine, PL-60781 Poznan, Poland b Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua, Via Gabelli 65, I-35121 Padua, Italy Received 31 January 2005; accepted 17 May 2005
Abstract Orexins A and B are hypothalamic peptides, that act via two subtypes of receptors, named OX1-R and OX2-R. Rat and human adrenal cortexes are provided with both OX1-R and OX2-R, and we have previously shown that orexin-A, but not orexin-B, enhances glucocorticoid secretion from dispersed adrenocortical cells. Since OX1-Rs preferentially bind orexin-A and OX2-Rs are non-selective for both orexins, the hypothesis has been advanced that the secretagogue effect of orexin-A is exclusively mediated by the OX1-R. Here, we aimed to verify this contention and to gain insight into the signaling mechanism(s) underlying the secretagogue effect of orexins using primary cultures of rat and human adrenocortical cells. Reverse transcription-polymerase chain reaction showed that cultured cells, as freshly dispersed cells, expressed both OX1-R and OX2-R mRNAs. Orexin-A, but not orexin-B, concentration-dependently increased corticosterone and cortisol secretion from cultured rat and human adrenocortical cells, respectively. The blockade of OX1-Rs by selective antibodies abrogated the secretagogue effect of orexin-A, while the immuno-blockade of OX2-Rs was ineffective. The glucocorticoid response of cultured cells to orexin-A was annulled by the adenylate cyclase and protein kinase (PK) A inhibitors SQ-22536 and H-89, and unaffected by the phospholipase C and PKC inhibitors U-73122 and calphostin-C. Orexin-A, but not orexin-B, enhanced cyclic-AMP production from cultured cells, and did not alter inositol-3-phosphate release. Collectively, our present results allow us to conclude that orexins stimulate glucocorticoid secretion from rat and human adrenocortical cells, exclusively acting through OX1-Rs coupled to the adenylate cyclase/PKA-dependent signaling cascade. © 2005 Elsevier Ltd. All rights reserved. Keywords: Orexins; Orexin receptors; Adrenocortical cells; Glucocorticoid secretion
1. Introduction Orexins A and B are two hypothalamic peptides involved in the central control of food intake [1,2] and sleep regulation [3,4]. They originate from the post-translational proteolytic processing of the prepro-orexin, and act via two subtypes of G protein-coupled receptors, referred to as OX1-R and OX2-R. The OX1-R preferentially binds orexin A, while the OX2-R is non-selective [5,6]. In recent years evidence has accumulated that orexins also play a role as neuroendocrine regulators (for review, ∗
Corresponding author. Tel.: +39 49 827 2317; fax: +39 49 827 2319. E-mail address: [email protected] (G.G. Nussdorfer).
0960-0760/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.05.003
see [7,8]). Accordingly, orexins, like other peptides involved in the central regulation of feeding (e.g. leptin and neuropeptide Y) [9–12], have been shown to influence the hypothalamic–pituitary–adrenal (HPA) axis (for review, see [13]). Moreover, orexins have been reported to act not only centrally in the HPA axis, but also peripherally, i.e. directly on the adrenal cortex. Consistent with this view, OX1-R and OX2-R expression has been detected in rat, pig and human adrenal cortex [13–20], and orexins have been found to stimulate glucocorticoid secretion from dispersed human and rat or cultured pig adrenocortical cells [20,21]. However, conflicting findings have been reported on the receptor subtype involved in the secretagogue action of orexins and their signaling
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mechanism: both OX1-R coupled to adenylate cyclase [17,20,21], and OX2-R coupled to both adenylate cyclase and inositol triphosphate (IP3) have been proposed as the major pathways [14,15,18]. It therefore seemed worthwhile to investigate the effects of orexins A and B on steroid secretion from rat and human adrenocortical cells in primary culture in vitro, and to try to address the subtype of receptor involved in the orexin action by selective immuno-blockade of OX1-R or OX2-R.
2. Materials and methods
On day 3 of culture, cells were incubated for 24 h as follows: (1) orexins A or B (10−10 , 10−8 or 10−6 M), and ACTH (10−8 M); (2) anti-OX1-R or anti-OX2-R antibody (5 g/mL) alone and in the presence of orexin A (10−6 M); (3) SQ-22536 (10−4 M), H-89 (10−5 M), U-73122 (10−5 M) or calphostin-C (10−5 M) alone and in the presence of orexin A (10−6 M). For each experiment, control cultures had no additions to the medium. In other experiments, cultures were incubated for 15 min in the presence of 10−6 M orexin A and orexin B or without any other addition. At the end of incubation, cells were counted in a CASY Cell Counter (Schaerfe System, Reutlingen, Germany), and medium was collected and stored at −80 ◦ C.
2.1. Reagents and adrenals Orexins A and B were purchased from Bachem (Bubendorf, Switzerland) and goat polyclonal anti-OX1-R (C-19) and anti-OX2-R (C-20) antibodies from Santa Cruz, (Santa Cruz, CA). The adenylate cyclase inhibitor SQ22536, the phospholipase C (PLC) inhibitor U-73122, the protein kinase (PK) A inhibitor H-89 and the PKC inhibitor calphostin-C (for references, see [22]) were obtained from BIOMOL Research Laboratories (Milan, Italy). Dulbecco’s modified minimum essential medium (DMEM), fetal calf serum (FCS), and all other chemicals were provided by Sigma–Aldrich Corporation (St. Louis, MO). Rat adrenals were obtained from adult female or male adult animals of the Wistar strain (160–200 g body weight), bred in our laboratory facilities. Human adrenal tails, which do not contain medullary chromaffin tissue [23] were obtained from six consenting male adult patients (48–60-years-old) undergoing unilateral nephrectomy–adrenalectomy for kidney cancer. The protocols of the experiments were approved by the local ethics committees for human and animal studies. 2.2. In vitro culture Rat adrenals and human adrenal tails were decapsulated to separate the zona glomerulosa, and rat glands were halved and enucleated to eliminate medullary chromaffin tissue. Dispersed zona fasciculata-reticularis cells were obtained by sequential collagenase digestion and mechanical disaggregation [23,24], and the purity of preparations (i.e. the lack of contaminant fibroblasts and endothelial cells) was routinarely checked by phase microscopy. Moreover, in some preparations immunocytochemistry demonstrated the absence of the typical stromal and endothelial markers prolyl-4-hydroxylase and von Willebrand factor, respectively. Dispersed cells were seeded at a density of 2 × 104 cells/cm2 into 24-well plates, and cultured for 48 h at 37 ◦ C in DMEM (with 1.125 g/L sodium bicarbonate, 10% FCS, 100 U/mL penicillin and 100 g/mL streptomycin added), and the medium changed every 24 h [25].
2.3. Reverse transcription (RT)-polymerase chain reaction (PCR) Adrenocortical cells were harvested from both dispersed cell and control culture dishes, and total RNA extracted and reverse transcribed to cDNA [26]. PCR was performed in a Delfi 100 thermal cycler (MJ Research Inc., Waterston, MA) following the procedures described earlier [27], using the previously published primers for human and rat OX1-Rs and OX2-Rs [28,29]. As a positive control, the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used. To rule out the possibility of amplifying genomic DNA, a control PCR was performed without prior RT of RNA. Detection of the PCR amplification product initially involved size fractionation on 2% agarose-gel electrophoresis; after purification using the QIA Quick PCR purification kit (Qiagen, Hilden, Germany), amplicons were identified by sequencing (Alf sequencer, Pharmacia Biotech, Freiburg, Germany). Primer sequence, predicted size of amplicons, and PCR programs are indicated in the legend of Fig. 1. The relative expression of OX1-R and OX2-R mRNAs in dispersed and cultured cells was assayed by real time-PCR in a Bio-Rad I-Cycler iQ Detection System (Bio-Rad Laboratories, Milan, Italy), using the following protocol: denaturation step (95 ◦ C for 3 min), 38 cycles of two steps of amplification (95 ◦ C for 15 s and annealing for 30 s), and melting curve (60–90 ◦ C with a heating rate of 0.5 ◦ C/10 s). 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. 2.4. Glucocorticoid assay Corticosterone and cortisol were extracted from culture media, and purified by HPLC [29]. Corticosterone concentration was measured by RIA, as previously described [30,31]. Sensitivity was 50 pg/mL, and intra- and interassay CVs 7.3 and 9.1%. Cortisol concentration was assayed by cortisol RIA
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experiments. Statistical comparison was by ANOVA, followed by Duncan’s multiple range test.
3. Results and discussion
Fig. 1. Ethidium bromide-stained 2% agarose gels showing cDNA amplified with OX1-R and OX2-R specific primers from RNA of exemplary dispersed (a) and cultured (b) rat (upper panel) and human adrenocortical cells (lower panel). Primer sequences were: (1) rat OX1-R: sense 5 -TGGGCTGTGTCGCTGGCTG-3 and antisense, 5 -GTTGGGGCTCTGTACACAGG-3 (amplicon, 328 bp); (2) rat OX2-R: sense, 5 -TTGGGGTTCACTGTCGTCAAG-3 and antisense, 5 -AGCCAGGTGGACAGGAGTGA3 (amplicon, 226 bp); (3) human OX1-R: sense, 5 -CCTTCCTGCCTGAAGTGAAG-3 and antisense, 5 -AGTGGGAGAAGGTGAAGCAG3 (amplicon, 189 bp); (4) human OX2-R: sense, 5 -ACATGGCACCACTGTGTCTC-3 and antisense, 5 -TGGCTCGGATCTGCTTTATT-3 (amplicon, 201 bp); (5) GAPDH: sense, 5 -CCCTCCATTGACCTCAACTA-3 and antisense, 5 -GCCAGTGAGCTTCCCGTTCA-3 (amplicon, 585 bp). The PCR programs were: (1) rat OX1-R and OX2-R, 35 cycles of 94 ◦ C for 60 s, 63 ◦ C for 60 s and 72 ◦ C for 60 s; (2) human OX1-R and OX2-R, 38 cycles of 95 ◦ C for 60 s, 59 ◦ C for 60 s and 72 ◦ C for 60 s. Lanes 1 were loaded with 200 ng of a size marker (Marker VIII; Roche, Mannheim, Germany). No amplification without prior RT of RNA is shown as a negative control.
(IRE-Sorin, Vercelli, Italy). Sensitivity was 30 pg/mL, and intra- and interassay CVs 6.5 and 8.2%. 2.5. cAMP and IP3 production For cAMP assay the phosphodiesterase inhibitor 3isobutyl-1-methylxanthine (10−4 M) was added to the incubation medium to prevent cAMP metabolism [32]. cAMP and IP3 were extracted and their concentrations measured by RIA, as described earlier [32], using commercial kits purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). The cAMP assay was Biotrack 432, with sensitivity of 1 pmol/L, and intra- and interassay CVs of 5.3 and 6.6%. The IP3 assay was Biotrack 1000, with sensitivity of 2 pmol/L, and intra- and interassay CVs of 6.5 and 8.1%. 2.6. Statistics Results were expressed as percent change from baseline values, and are shown as the mean ± S.E.M. of six separate
RT-PCR showed expression of OX1-R and OX2-R mRNA in both rat and human cultured adrenocortical cells, as well as in freshly dispersed cell preparations (Fig. 1). Real time-PCR did not reveal major differences in the level of expression of both receptors between dispersed and cultured cells (Table 1). These findings confirm that rat and human adrenocortical cells express both orexin-receptor subtypes [16,17,33], and not predominantly OX2-R, as previously suggested [13–15,18]. It is difficult to explain these discrepancies, although it should be taken into account that the predominance of OX2-R expression has been observed by examining the RNA extracted from the entire adrenal, i.e. containing cortex and medulla. Moreover, we showed that 48 h of in vitro culture does not alter orexin receptor expression in adrenocortical cells, which indicates that primary cultures are a suitable model to study the effect of orexins on adrenal-cortex functions. Orexin-A raised glucocorticoid secretion from cultured rat and human adrenocortical cells in a dose-related fashion. Minimal effective concentrations were 10−8 M for the corticosterone response from rat cells and 10−10 M for cortisol from human cells, with the secretagogue effect of 10−6 M orexin-A about 50% that of 10−8 M ACTH. Orexin-B did not elicit any secretory response (Fig. 2). Evidence has been provided that orexin-B is selective for OX2-R (EC50 of 60 nM for OX2-R and 2500 nM for OX1-R), while orexin-A binds with similar potency both receptor subtypes (EC50 of 30 nM for OX1-R and 34 nM for OX2-R) [5,6,13]. Hence, our observations are consistent with the view that the secretagogue action of orexins is mediated via the OX1-R [17,21]. This contention is further supported by the demonstration that blockade with selective antibodies of OX1-R but not OX2-R abolished the secretory response of cultured cells to 10−6 M orexin-A (Fig. 3). Our investigation also provides insight into the signaling cascades involved in the OX1-R-mediated secretagogue action of orexin-A. Both the adenylate cyclase inhibitor SQ-22536 [34] and the PKA inhibitor H-89 [35] blocked Table 1 Relative expression of OX1-R and OX2-R mRNA in dispersed and cultured rat and human adrenocortical cells Target gene/GAPDH ratio
OX1-R
OX2-R
Rats Dispersed cells Cultured cells
0.84 ± 0.15 0.75 ± 0.16
0.68 ± 0.14 0.78 ± 0.15
Humans Dispersed cells Cultured cells
0.92 ± 0.30 1.12 ± 0.32
1.06 ± 0.28 0.94 ± 0.25
Data are mean ± S.D. of four separate experiments.
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Fig. 3. Effects of OX1-R (left panels) and OX2-R immunoblockade (right panels) on basal and orexin-A (10−6 M)-stimulated glucocorticoid secretion from cultured rat (upper panel) and human adrenocortical cells (lower panel). Data are expressed as percent change from the respective baseline control value (taken equal to 100), and are mean ± S.E.M. (n = 6). ** P < 0.01 from the respective baseline value; a P < 0.01 from the respective control value. Fig. 2. Effects of orexin-A (left panels) and orexin-B (right panels) on corticosterone secretion from cultured rat (upper panel) and cortisol secretion from cultured human adrenocortical cells (lower panel). The effect of ACTH (10−8 M) is shown as a positive control. Data are expressed as percent change from control (taken equal to 100), and are mean ± S.E.M. (n = 6). * P < 0.05 and ** P < 0.01 from the respective baseline (B) value.
the glucocorticoid secretory response of rat and human cultured cells to 10−6 M orexin-A, while the exposure to either the PLC inhibitor U-73122 [36] or the PKC inhibitor calphostin-C [37] was ineffective (Fig. 4). A non-specific toxic effect of SQ-22536 and H-89 on the steroidogenic machinery appears unlikely in that these inhibitors did not alter basal glucocorticoid secretion from cultured cells (Fig. 4). Taken together, these findings strongly suggest that
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Fig. 5. Effects of orexin-A and orexin-B (10−6 M) on basal cAMP and IP3 production from cultured rat (right panel) and human adrenocortical cells (left panel). Data are expressed as percent change from the respective baseline value (taken equal to 100), and are mean ± S.E.M. (n = 6). ** P < 0.01 from the respective baseline value.
Fig. 4. Effects of the inhibitors of adenylate cyclase (SQ-22536), PKA (H-89), PLC (U-73122) and PKC (calphostin-C) on basal and orexin-A (10−6 M)-stimulated glucocorticoid secretion from cultured rat (upper panel) and human adrenocortical cells (lower panel). Data are expressed as percent change from the respective baseline control value (taken equal to 100), and are mean ± S.E.M. (n = 6). ** P < 0.01 from the respective baseline value; a P < 0.01 from the respective control value.
orexins stimulate glucocorticoid secretion from cultured adrenocortical cells through OX1-R coupled to an adenylate cyclase/PKA-dependent cascade. This contention is further supported by the demonstration that 10−6 M orexin-A enhanced cAMP, but not IP3, production from cultured cells, while orexin-B did not evoke any response (Fig. 5). The physiological relevance of our findings remains to be ascertained, especially as far as rats are concerned where orexin-A is far less effective than in humans. Under basal conditions the blood levels of orexin-A in rat (15 pM) and
human healthy volunteers (2 pM) [38,39] are about two orders of magnitude lower than the minimal effective concentration of orexin-A eliciting an in vitro secretory response from rat and human adrenocortical cells (10−8 and 10−10 M, respectively), which rules out the possibility that orexins act systemically on the adrenals, at least under normal conditions. However, orexin levels increase in fasted hypoglycemic animals [1,2], so that they may take part, along with leptin (which is down-regulated in obese and overfed animals [40], and inhibit glucocorticoid secretion [41,42]), in the maintenance of normoglycemia and body-weight homeostasis under conditions of feeding dysregulation. Moreover, many lines of evidence indicate that various peptides which are able to modulate steroid-hormone secretion act in an autocrine–paracrine manner, being synthesized in the adrenal cortex and medulla (for review, see [43]). Although neither prepro-orexin mRNA expression has been detected in rat adrenals [29,44] nor orexin-A immunoreactivity in human glands [45], prepro-orexin and orexin-A expression has been demonstrated by RT-PCR and Western blotting in human adrenals [15,18,46] and by RIA in human cortisol-secreting adrenocortical adenomas [33]. Hence, the possibility that orexins may act as autocrine–paracrine regulators of adrenal secretion in humans merits further exploration. Finally, OX2-R are highly expressed in rat and human adrenocortical cells. Previous studies have shown that OX2-R are involved in the action of orexins in modulating the secretory activity of rat PC12 cells [47] and of human pheochromocytomas [48], but our present results make it unlike that this receptor subtype takes part in the adrenocortical secretagogue action of orexins. The question remains of whether OX2-R are silent receptors, or whether they may be linked to a possible growth promoting effect of orexins in the adrenal
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cortex. The latter possibility appears to be the most convincing because both orexin-A and orexin-B were found to enhance the proliferative activity of cultured human adrenal adenoma cells [33]. In conclusion, our study provides novel evidence that, although cultured rat and human adrenocortical cells equally express OX1-R and OX2-R, only OX1-R coupled to the adenylate cyclase/PKA-dependent signaling cascade is involved in the glucocorticoid secretagogue action of orexins.
Acknowledgments
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[38] Z. Arihara, K. Takahashi, O. Murakami, K. Totsune, M. Sone, F. Satoh, S. Ito, T. Mouri, Immunoreactive orexin-A in human plasma, Peptides 22 (2001) 139–142. [39] O. J¨ohren, N. Br¨uggemann, A. Dendorfer, P. Dominiak, Gonadal steroids differentially regulate the messenger ribonucleic acid expression of pituitary orexin type 1 receptors and adrenal orexin type 2 receptors, Endocrinology 144 (2003) 1219–1225. [40] R.S. Ahima, J.S. Flier, Leptin, Annu. Rev. Physiol. 62 (2000) 413–437. [41] S.R. Bornstein, K. Uhlmann, A. Haidan, M. Ehrhart-Bornstein, W.A. Scherbaum, Evidence for a novel peripheral action of leptin as a metabolic signal to adrenal gland. Leptin inhibits cortisol release directly, Diabetes 46 (1997) 1235–1238. [42] A. Glasow, A. Haidan, U. Hilbers, M. Breidert, J. Gilliespie, W.A. Scherbaum, G.P. Chrousos, S.R. Bornstein, Expression of Ob receptor in normal human adrenals: differential regulation of adrenocortical and adrenomedullary function by leptin, J. Clin. Endocrinol. Metab. 83 (1998) 4459–4466. [43] G.G. Nussdorfer, Paracrine control of adrenal cortical function by medullary chromaffin cells, Pharmacol. Rev. 48 (1996) 495–530. [44] M. Lopez, L. Seoane, R.M. Se˜naris, C. Dieguez, Prepro-orexin mRNA levels in the rat hypothalamus, and orexin receptor m-RNA levels in the rat hypothalamus and adrenal gland are not influenced by the thyroid status, Neurosci. Lett. 300 (2001) 171–175. [45] M. Nakabayashi, T. Suzuki, K. Takahashi, K. Totsune, Y. Muramatsu, C. Kaneko, F. Date, J. Takeyama, A.D. Darnel, T. Moriya, H. Sasano, Orexin-A expression in human peripheral tissues, Mol. Cell. Endocrinol. 205 (2003) 43–50. [46] Z. Arihara, K. Takahashi, O. Murakami, K. Totsune, M. Sone, F. Satoh, S. Ito, Y. Hayashi, H. Sasano, T. Mouri, Orexin-A in the human brain and tumor tissues of ganglioneuroblastoma and neuroblastoma, Peptides 21 (2000) 565–570. [47] T. Nanmoku, K. Isobe, T. Sakurai, A. Yamanaka, T. Takekoshi, Y. Kawakami, K. Ishii, K. Goto, T. Nakai, Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells, Biochem. Biophys. Res. Commun. 277 (2000) 310–315. [48] G. Mazzocchi, L.K. Malendowicz, F. Aragona, P. Rebuffat, L. Gottardo, G.G. Nussdorfer, Human pheochromocytomas express orexin receptor type 2 gene and display an in vitro secretory response to orexins A and B, J. Clin. Endocrinol. Metab. 86 (2001) 4818–4821.
Orexins stimulate glucocorticoid secretion from cultured rat and human adrenocortical cells, exclusively acting via the OX1 receptor Agnieszka Ziolkowska a , Raffaella Spinazzi b , Giovanna Albertin b , Magdalena Nowak a , Ludwik K. Malendowicz a , Cinzia Tortorella b , Gastone G. Nussdorfer b,∗ a
Department of Histology and Embryology, Poznan School of Medicine, PL-60781 Poznan, Poland b Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua, Via Gabelli 65, I-35121 Padua, Italy Received 31 January 2005; accepted 17 May 2005
Abstract Orexins A and B are hypothalamic peptides, that act via two subtypes of receptors, named OX1-R and OX2-R. Rat and human adrenal cortexes are provided with both OX1-R and OX2-R, and we have previously shown that orexin-A, but not orexin-B, enhances glucocorticoid secretion from dispersed adrenocortical cells. Since OX1-Rs preferentially bind orexin-A and OX2-Rs are non-selective for both orexins, the hypothesis has been advanced that the secretagogue effect of orexin-A is exclusively mediated by the OX1-R. Here, we aimed to verify this contention and to gain insight into the signaling mechanism(s) underlying the secretagogue effect of orexins using primary cultures of rat and human adrenocortical cells. Reverse transcription-polymerase chain reaction showed that cultured cells, as freshly dispersed cells, expressed both OX1-R and OX2-R mRNAs. Orexin-A, but not orexin-B, concentration-dependently increased corticosterone and cortisol secretion from cultured rat and human adrenocortical cells, respectively. The blockade of OX1-Rs by selective antibodies abrogated the secretagogue effect of orexin-A, while the immuno-blockade of OX2-Rs was ineffective. The glucocorticoid response of cultured cells to orexin-A was annulled by the adenylate cyclase and protein kinase (PK) A inhibitors SQ-22536 and H-89, and unaffected by the phospholipase C and PKC inhibitors U-73122 and calphostin-C. Orexin-A, but not orexin-B, enhanced cyclic-AMP production from cultured cells, and did not alter inositol-3-phosphate release. Collectively, our present results allow us to conclude that orexins stimulate glucocorticoid secretion from rat and human adrenocortical cells, exclusively acting through OX1-Rs coupled to the adenylate cyclase/PKA-dependent signaling cascade. © 2005 Elsevier Ltd. All rights reserved. Keywords: Orexins; Orexin receptors; Adrenocortical cells; Glucocorticoid secretion
1. Introduction Orexins A and B are two hypothalamic peptides involved in the central control of food intake [1,2] and sleep regulation [3,4]. They originate from the post-translational proteolytic processing of the prepro-orexin, and act via two subtypes of G protein-coupled receptors, referred to as OX1-R and OX2-R. The OX1-R preferentially binds orexin A, while the OX2-R is non-selective [5,6]. In recent years evidence has accumulated that orexins also play a role as neuroendocrine regulators (for review, ∗
Corresponding author. Tel.: +39 49 827 2317; fax: +39 49 827 2319. E-mail address: [email protected] (G.G. Nussdorfer).
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see [7,8]). Accordingly, orexins, like other peptides involved in the central regulation of feeding (e.g. leptin and neuropeptide Y) [9–12], have been shown to influence the hypothalamic–pituitary–adrenal (HPA) axis (for review, see [13]). Moreover, orexins have been reported to act not only centrally in the HPA axis, but also peripherally, i.e. directly on the adrenal cortex. Consistent with this view, OX1-R and OX2-R expression has been detected in rat, pig and human adrenal cortex [13–20], and orexins have been found to stimulate glucocorticoid secretion from dispersed human and rat or cultured pig adrenocortical cells [20,21]. However, conflicting findings have been reported on the receptor subtype involved in the secretagogue action of orexins and their signaling
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mechanism: both OX1-R coupled to adenylate cyclase [17,20,21], and OX2-R coupled to both adenylate cyclase and inositol triphosphate (IP3) have been proposed as the major pathways [14,15,18]. It therefore seemed worthwhile to investigate the effects of orexins A and B on steroid secretion from rat and human adrenocortical cells in primary culture in vitro, and to try to address the subtype of receptor involved in the orexin action by selective immuno-blockade of OX1-R or OX2-R.
2. Materials and methods
On day 3 of culture, cells were incubated for 24 h as follows: (1) orexins A or B (10−10 , 10−8 or 10−6 M), and ACTH (10−8 M); (2) anti-OX1-R or anti-OX2-R antibody (5 g/mL) alone and in the presence of orexin A (10−6 M); (3) SQ-22536 (10−4 M), H-89 (10−5 M), U-73122 (10−5 M) or calphostin-C (10−5 M) alone and in the presence of orexin A (10−6 M). For each experiment, control cultures had no additions to the medium. In other experiments, cultures were incubated for 15 min in the presence of 10−6 M orexin A and orexin B or without any other addition. At the end of incubation, cells were counted in a CASY Cell Counter (Schaerfe System, Reutlingen, Germany), and medium was collected and stored at −80 ◦ C.
2.1. Reagents and adrenals Orexins A and B were purchased from Bachem (Bubendorf, Switzerland) and goat polyclonal anti-OX1-R (C-19) and anti-OX2-R (C-20) antibodies from Santa Cruz, (Santa Cruz, CA). The adenylate cyclase inhibitor SQ22536, the phospholipase C (PLC) inhibitor U-73122, the protein kinase (PK) A inhibitor H-89 and the PKC inhibitor calphostin-C (for references, see [22]) were obtained from BIOMOL Research Laboratories (Milan, Italy). Dulbecco’s modified minimum essential medium (DMEM), fetal calf serum (FCS), and all other chemicals were provided by Sigma–Aldrich Corporation (St. Louis, MO). Rat adrenals were obtained from adult female or male adult animals of the Wistar strain (160–200 g body weight), bred in our laboratory facilities. Human adrenal tails, which do not contain medullary chromaffin tissue [23] were obtained from six consenting male adult patients (48–60-years-old) undergoing unilateral nephrectomy–adrenalectomy for kidney cancer. The protocols of the experiments were approved by the local ethics committees for human and animal studies. 2.2. In vitro culture Rat adrenals and human adrenal tails were decapsulated to separate the zona glomerulosa, and rat glands were halved and enucleated to eliminate medullary chromaffin tissue. Dispersed zona fasciculata-reticularis cells were obtained by sequential collagenase digestion and mechanical disaggregation [23,24], and the purity of preparations (i.e. the lack of contaminant fibroblasts and endothelial cells) was routinarely checked by phase microscopy. Moreover, in some preparations immunocytochemistry demonstrated the absence of the typical stromal and endothelial markers prolyl-4-hydroxylase and von Willebrand factor, respectively. Dispersed cells were seeded at a density of 2 × 104 cells/cm2 into 24-well plates, and cultured for 48 h at 37 ◦ C in DMEM (with 1.125 g/L sodium bicarbonate, 10% FCS, 100 U/mL penicillin and 100 g/mL streptomycin added), and the medium changed every 24 h [25].
2.3. Reverse transcription (RT)-polymerase chain reaction (PCR) Adrenocortical cells were harvested from both dispersed cell and control culture dishes, and total RNA extracted and reverse transcribed to cDNA [26]. PCR was performed in a Delfi 100 thermal cycler (MJ Research Inc., Waterston, MA) following the procedures described earlier [27], using the previously published primers for human and rat OX1-Rs and OX2-Rs [28,29]. As a positive control, the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used. To rule out the possibility of amplifying genomic DNA, a control PCR was performed without prior RT of RNA. Detection of the PCR amplification product initially involved size fractionation on 2% agarose-gel electrophoresis; after purification using the QIA Quick PCR purification kit (Qiagen, Hilden, Germany), amplicons were identified by sequencing (Alf sequencer, Pharmacia Biotech, Freiburg, Germany). Primer sequence, predicted size of amplicons, and PCR programs are indicated in the legend of Fig. 1. The relative expression of OX1-R and OX2-R mRNAs in dispersed and cultured cells was assayed by real time-PCR in a Bio-Rad I-Cycler iQ Detection System (Bio-Rad Laboratories, Milan, Italy), using the following protocol: denaturation step (95 ◦ C for 3 min), 38 cycles of two steps of amplification (95 ◦ C for 15 s and annealing for 30 s), and melting curve (60–90 ◦ C with a heating rate of 0.5 ◦ C/10 s). 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. 2.4. Glucocorticoid assay Corticosterone and cortisol were extracted from culture media, and purified by HPLC [29]. Corticosterone concentration was measured by RIA, as previously described [30,31]. Sensitivity was 50 pg/mL, and intra- and interassay CVs 7.3 and 9.1%. Cortisol concentration was assayed by cortisol RIA
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experiments. Statistical comparison was by ANOVA, followed by Duncan’s multiple range test.
3. Results and discussion
Fig. 1. Ethidium bromide-stained 2% agarose gels showing cDNA amplified with OX1-R and OX2-R specific primers from RNA of exemplary dispersed (a) and cultured (b) rat (upper panel) and human adrenocortical cells (lower panel). Primer sequences were: (1) rat OX1-R: sense 5 -TGGGCTGTGTCGCTGGCTG-3 and antisense, 5 -GTTGGGGCTCTGTACACAGG-3 (amplicon, 328 bp); (2) rat OX2-R: sense, 5 -TTGGGGTTCACTGTCGTCAAG-3 and antisense, 5 -AGCCAGGTGGACAGGAGTGA3 (amplicon, 226 bp); (3) human OX1-R: sense, 5 -CCTTCCTGCCTGAAGTGAAG-3 and antisense, 5 -AGTGGGAGAAGGTGAAGCAG3 (amplicon, 189 bp); (4) human OX2-R: sense, 5 -ACATGGCACCACTGTGTCTC-3 and antisense, 5 -TGGCTCGGATCTGCTTTATT-3 (amplicon, 201 bp); (5) GAPDH: sense, 5 -CCCTCCATTGACCTCAACTA-3 and antisense, 5 -GCCAGTGAGCTTCCCGTTCA-3 (amplicon, 585 bp). The PCR programs were: (1) rat OX1-R and OX2-R, 35 cycles of 94 ◦ C for 60 s, 63 ◦ C for 60 s and 72 ◦ C for 60 s; (2) human OX1-R and OX2-R, 38 cycles of 95 ◦ C for 60 s, 59 ◦ C for 60 s and 72 ◦ C for 60 s. Lanes 1 were loaded with 200 ng of a size marker (Marker VIII; Roche, Mannheim, Germany). No amplification without prior RT of RNA is shown as a negative control.
(IRE-Sorin, Vercelli, Italy). Sensitivity was 30 pg/mL, and intra- and interassay CVs 6.5 and 8.2%. 2.5. cAMP and IP3 production For cAMP assay the phosphodiesterase inhibitor 3isobutyl-1-methylxanthine (10−4 M) was added to the incubation medium to prevent cAMP metabolism [32]. cAMP and IP3 were extracted and their concentrations measured by RIA, as described earlier [32], using commercial kits purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). The cAMP assay was Biotrack 432, with sensitivity of 1 pmol/L, and intra- and interassay CVs of 5.3 and 6.6%. The IP3 assay was Biotrack 1000, with sensitivity of 2 pmol/L, and intra- and interassay CVs of 6.5 and 8.1%. 2.6. Statistics Results were expressed as percent change from baseline values, and are shown as the mean ± S.E.M. of six separate
RT-PCR showed expression of OX1-R and OX2-R mRNA in both rat and human cultured adrenocortical cells, as well as in freshly dispersed cell preparations (Fig. 1). Real time-PCR did not reveal major differences in the level of expression of both receptors between dispersed and cultured cells (Table 1). These findings confirm that rat and human adrenocortical cells express both orexin-receptor subtypes [16,17,33], and not predominantly OX2-R, as previously suggested [13–15,18]. It is difficult to explain these discrepancies, although it should be taken into account that the predominance of OX2-R expression has been observed by examining the RNA extracted from the entire adrenal, i.e. containing cortex and medulla. Moreover, we showed that 48 h of in vitro culture does not alter orexin receptor expression in adrenocortical cells, which indicates that primary cultures are a suitable model to study the effect of orexins on adrenal-cortex functions. Orexin-A raised glucocorticoid secretion from cultured rat and human adrenocortical cells in a dose-related fashion. Minimal effective concentrations were 10−8 M for the corticosterone response from rat cells and 10−10 M for cortisol from human cells, with the secretagogue effect of 10−6 M orexin-A about 50% that of 10−8 M ACTH. Orexin-B did not elicit any secretory response (Fig. 2). Evidence has been provided that orexin-B is selective for OX2-R (EC50 of 60 nM for OX2-R and 2500 nM for OX1-R), while orexin-A binds with similar potency both receptor subtypes (EC50 of 30 nM for OX1-R and 34 nM for OX2-R) [5,6,13]. Hence, our observations are consistent with the view that the secretagogue action of orexins is mediated via the OX1-R [17,21]. This contention is further supported by the demonstration that blockade with selective antibodies of OX1-R but not OX2-R abolished the secretory response of cultured cells to 10−6 M orexin-A (Fig. 3). Our investigation also provides insight into the signaling cascades involved in the OX1-R-mediated secretagogue action of orexin-A. Both the adenylate cyclase inhibitor SQ-22536 [34] and the PKA inhibitor H-89 [35] blocked Table 1 Relative expression of OX1-R and OX2-R mRNA in dispersed and cultured rat and human adrenocortical cells Target gene/GAPDH ratio
OX1-R
OX2-R
Rats Dispersed cells Cultured cells
0.84 ± 0.15 0.75 ± 0.16
0.68 ± 0.14 0.78 ± 0.15
Humans Dispersed cells Cultured cells
0.92 ± 0.30 1.12 ± 0.32
1.06 ± 0.28 0.94 ± 0.25
Data are mean ± S.D. of four separate experiments.
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Fig. 3. Effects of OX1-R (left panels) and OX2-R immunoblockade (right panels) on basal and orexin-A (10−6 M)-stimulated glucocorticoid secretion from cultured rat (upper panel) and human adrenocortical cells (lower panel). Data are expressed as percent change from the respective baseline control value (taken equal to 100), and are mean ± S.E.M. (n = 6). ** P < 0.01 from the respective baseline value; a P < 0.01 from the respective control value. Fig. 2. Effects of orexin-A (left panels) and orexin-B (right panels) on corticosterone secretion from cultured rat (upper panel) and cortisol secretion from cultured human adrenocortical cells (lower panel). The effect of ACTH (10−8 M) is shown as a positive control. Data are expressed as percent change from control (taken equal to 100), and are mean ± S.E.M. (n = 6). * P < 0.05 and ** P < 0.01 from the respective baseline (B) value.
the glucocorticoid secretory response of rat and human cultured cells to 10−6 M orexin-A, while the exposure to either the PLC inhibitor U-73122 [36] or the PKC inhibitor calphostin-C [37] was ineffective (Fig. 4). A non-specific toxic effect of SQ-22536 and H-89 on the steroidogenic machinery appears unlikely in that these inhibitors did not alter basal glucocorticoid secretion from cultured cells (Fig. 4). Taken together, these findings strongly suggest that
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Fig. 5. Effects of orexin-A and orexin-B (10−6 M) on basal cAMP and IP3 production from cultured rat (right panel) and human adrenocortical cells (left panel). Data are expressed as percent change from the respective baseline value (taken equal to 100), and are mean ± S.E.M. (n = 6). ** P < 0.01 from the respective baseline value.
Fig. 4. Effects of the inhibitors of adenylate cyclase (SQ-22536), PKA (H-89), PLC (U-73122) and PKC (calphostin-C) on basal and orexin-A (10−6 M)-stimulated glucocorticoid secretion from cultured rat (upper panel) and human adrenocortical cells (lower panel). Data are expressed as percent change from the respective baseline control value (taken equal to 100), and are mean ± S.E.M. (n = 6). ** P < 0.01 from the respective baseline value; a P < 0.01 from the respective control value.
orexins stimulate glucocorticoid secretion from cultured adrenocortical cells through OX1-R coupled to an adenylate cyclase/PKA-dependent cascade. This contention is further supported by the demonstration that 10−6 M orexin-A enhanced cAMP, but not IP3, production from cultured cells, while orexin-B did not evoke any response (Fig. 5). The physiological relevance of our findings remains to be ascertained, especially as far as rats are concerned where orexin-A is far less effective than in humans. Under basal conditions the blood levels of orexin-A in rat (15 pM) and
human healthy volunteers (2 pM) [38,39] are about two orders of magnitude lower than the minimal effective concentration of orexin-A eliciting an in vitro secretory response from rat and human adrenocortical cells (10−8 and 10−10 M, respectively), which rules out the possibility that orexins act systemically on the adrenals, at least under normal conditions. However, orexin levels increase in fasted hypoglycemic animals [1,2], so that they may take part, along with leptin (which is down-regulated in obese and overfed animals [40], and inhibit glucocorticoid secretion [41,42]), in the maintenance of normoglycemia and body-weight homeostasis under conditions of feeding dysregulation. Moreover, many lines of evidence indicate that various peptides which are able to modulate steroid-hormone secretion act in an autocrine–paracrine manner, being synthesized in the adrenal cortex and medulla (for review, see [43]). Although neither prepro-orexin mRNA expression has been detected in rat adrenals [29,44] nor orexin-A immunoreactivity in human glands [45], prepro-orexin and orexin-A expression has been demonstrated by RT-PCR and Western blotting in human adrenals [15,18,46] and by RIA in human cortisol-secreting adrenocortical adenomas [33]. Hence, the possibility that orexins may act as autocrine–paracrine regulators of adrenal secretion in humans merits further exploration. Finally, OX2-R are highly expressed in rat and human adrenocortical cells. Previous studies have shown that OX2-R are involved in the action of orexins in modulating the secretory activity of rat PC12 cells [47] and of human pheochromocytomas [48], but our present results make it unlike that this receptor subtype takes part in the adrenocortical secretagogue action of orexins. The question remains of whether OX2-R are silent receptors, or whether they may be linked to a possible growth promoting effect of orexins in the adrenal
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cortex. The latter possibility appears to be the most convincing because both orexin-A and orexin-B were found to enhance the proliferative activity of cultured human adrenal adenoma cells [33]. In conclusion, our study provides novel evidence that, although cultured rat and human adrenocortical cells equally express OX1-R and OX2-R, only OX1-R coupled to the adenylate cyclase/PKA-dependent signaling cascade is involved in the glucocorticoid secretagogue action of orexins.
Acknowledgments
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[17]
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We thank Miss Alberta Coi for her invaluable help in the search and delivery of bibliographic items. [19]
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