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Human Pheochromocytomas Express Orexin Receptor Type 2 Gene and Display an in Vitro Secretory Response to Orexins A and B

Departments of Human Anatomy and Physiology, Section of Anatomy (G.M., L.K.M., P.R., L.G., G.G.N.), and Urology (F.A.), University of Padua, I-35121 Padua, Italy

Address all correspondence and requests for reprints to: Prof. G. Mazzocchi, Department of Human Anatomy and Physiology, Section of Anatomy, Via Gabelli 65, I-35121 Padova, Italy. E-mail: mazzocch{at}ux1.unipd.it

Abstract

Orexins A and B are hypothalamic peptides, that act through two receptor subtypes, called OX1-R and OX2-R. OX1-R selectively binds orexin A, whereas OX2-R is nonselective for both orexins. High levels of OX1-R mRNA and low levels of OX2-R mRNA have been previously detected in the human adrenal cortex and medulla. Here we demonstrated by RT-PCR the expression of the OX2-R, but not the OX1-R, gene in 10 benign secreting pheochromocytomas. Both orexins A and B stimulated catecholamine secretion from pheochromocytoma slices; the maximal effective concentration was 10^-8 mol/liter. Orexins A and B (10^-8 mol/liter) increased IP3, but not cAMP production, by tumor slices, and the effect was blocked by the PLC inhibitor U-73122. The catecholamine response to 10^-8 mol/liter orexins A and B was abolished by either U-73122 or the PKC antagonist calphostin C and was unaffected by the adenylate cyclase inhibitor SQ-22536 and the PKA inhibitor H-89. Collectively, these findings suggest that orexins stimulate catecholamine secretion from human pheochromocytomas, acting through OX2-R coupled to the PLC-PKC signaling pathway.

OREXINS A AND B are two hypothalamic peptides that play a role in the central control of food intake (1, 2, 3, 4) and perhaps in sleep regulation (5, 6). They originate from the posttranslational proteolytic cleavage of a common precursor, the prepro-orexin, and act via two subtypes of G protein-coupled receptors, referred to as OX1-R and OX2-R (1, 7).

Evidence indicates that orexins, in addition to their central functions, exert peripheral effects, the adrenal gland being one of the target organs. Orexins were found to stimulate corticosterone secretion from rat adrenal cortex both in vivo and in vitro (8) and cortisol production from dispersed human adrenocortical cells, acting through the OX1-R (9). Accordingly, high levels of OX1-R mRNA were detected by RT-PCR in the human adrenal zona fasciculata-reticularis (9). In contrast, investigations dealing with the effects of orexins on adrenal catecholamine secretion gave conflicting results. Although human and rat adrenal chromaffin cells were found to express OX1-R and OX2-R mRNAs (9, 10), orexins did not apparently affect catecholamine release by human (9) and rat adrenomedullary slices (Mazzocchi, G., and G. G. Nussdorfer, unpublished data). In contrast, orexins were reported to inhibit catecholamine synthesis and secretion from rat PC12 cells (11).

Human benign pheochromocytomas are adrenal medulla-derived tumors that display a great variability (12) and are known to express several regulatory peptides and their receptors (13). Hence, the present study was designed to investigate whether pheochromocytomas express orexin receptor genes and possess a secretory response to orexins.

Subjects and Methods

Patients

Ten patients with unilateral intraadrenal pheochromocytomas were recruited. They exhibited a positive response to a glucagon provocative test and displayed high basal levels of plasma and urinary catecholamines. The patients underwent surgery, and resected tumors were histologically identified as well differentiated benign pheochromocytomas. The patients gave written consent, and the study protocol was approved by the local ethics committee for human studies.

RT-PCR

Fragments of each pheochromocytoma 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 the cell biology laboratory. RNA was extracted with the guanidium isothiocyanate method, and 1 µg total RNA was reverse transcribed to cDNA, as previously detailed (14). The 5'- and 3'-primers were selected with Oligo 3.0 primer analysis software on the basis of GenBank cDNA sequences of human origin (1): the primer sequences and the size of the amplification products are shown in Table 1. As a positive control, amplification of a fragment of the human glyceraldehyde-3-phosphate dehydrogenase mRNA was performed, using up- and downstream primers selected according to the method described by Tso et al. (15) (Table 1). To rule out the possibility of amplifying genomic DNA, in some experiments PCR was performed without prior RT of the RNA. In a Delphi 100 Thermal Cycler (Oracle Biosystem, MJ Research, Inc., Waterston, MA), we used a denaturation step at 95 C for 1 min, an annealing step at 59 C for 1 min, and an extension step at 72 C for 1 min for a total of 38 cycles. An additional extension step at 72 for 7 min was then performed. Detection of the PCR amplification products was first carried out by size fractionation on 2% agarose gel electrophoresis. After purification using the QIAQuick PCR purification kit (QIAGEN, Hilden, Germany), PCR products were identified by sequencing on an Alf sequencer (Pharmacia Biotech, Freiburg, Germany).

Pheochromocytoma slices were put in medium 199 (Difco, Detroit, MI) and Krebs-Ringer bicarbonate buffer with 2% glucose, containing 5 mg/ml human serum albumin (Sigma, St. Louis, MO). Slices were incubated (3 mg/ml, in replicates of three each) as follows: 1) orexins A or B (Peninsula Laboratories, Inc., St. Helens, UK) from 10^-10–10^-6 mol/liter (pheochromocytomas 1–4), and 2) orexins A or B (10^-8 mol/liter) alone and in the presence of 10^-4 mol/liter SQ-22536, 10^-5 mol/liter H-89, 10^-5 mol/liter U-73122, or 10^-5 mol/liter calphostin C (BIOMOL Research Laboratories, Inc., Milan, Italy; pheochromocytomas 5–10). In the case of cAMP assay (see below), 10^-4 mol/liter isobutylmethylxanthine (Sigma), a phosphodiesterase inhibitor, was added to prevent cAMP metabolism. Incubation was carried out for 15 min in a shaking bath at 37 C in an atmosphere of 95% air-5% CO₂.

Catecholamine, cAMP, and IP3 assays

The catecholamine concentration in the incubation medium was measured by HPLC, using a reverse phase column and a glassy carbon electrochemical detector (16). The sensitivity of the assay was 3 pmol/liter, and intra- and interassay coefficients of variation were 6.5% and 7.6%, respectively.

cAMP was extracted by incubating the medium with 0.1 N HCl for 20 min at 4 C. The HCl extract was then neutralized, and the cAMP concentration was determined using Biotrak TRK 432 (Amersham Pharmacia Biotech, Little Chalfont, UK). The sensitivity of the assay was 1 pmol/liter, and intra- and interassay coefficients of variation were 5.3% and 6.6%, respectively.

IP3 was extracted by the trichloroacetic acid method and purified by Amprex SAX-minicolumn chromatography, and its concentration was measured by RIA. The procedure followed the protocol of the Biotrak TRK 1000 (Amersham Pharmacia Biotech). The sensitivity of the assay was 2 pmol/liter, and intra- and interassay coefficients of variation were 6.8% and 8.1%, respectively.

The protein concentration of the incubated tissue fragments was determined by the Lowry method, using BSA (Sigma) as a standard, and catecholamine, cAMP, and IP3 productions were expressed as picomoles per mg protein.

Statistics

Data obtained from each pheochromocytoma were averaged and expressed as the mean ± SEM of four or six separate experiments (four or six pheochromocytomas from four or six patients). The statistical comparison of results was performed by ANOVA, followed by Duncan’s multiple range test.

Results

RT-PCR demonstrated the presence of high levels of OX2-R mRNA in the 10 pheochromocytomas. Conversely, OX1-R gene expression was absent (Fig. 1).

View larger version (68K): [in this window] [in a new window]   Figure 1. Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human OX1-R-, OX2-R-, and glyceraldehyde-3-phosphate dehydrogenase-specific primers from RNA of a series of 10 pheochromocytomas. Lane 1 was loaded with 200 ng of a size marker (SM VIII or SM XIV, Roche Molecular Biochemicals, Indianapolis, IN). Only amplified fragments of the size corresponding to OX2-R were detected: 201 bp (primer a) and 600 bp (primer b). No amplification with water instead of RNA, as a negative control, is shown.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effects of orexins on epinephrine (left panel) and norepinephrine release (right panel) by pheochromocytoma slices. The means ± SEM of four separate experiments (pheochromocytomas 1–4) are shown. *, P < 0.01 vs. the respective baseline value.

View larger version (35K): [in this window] [in a new window]   Figure 3. Effects of orexins (10-8 mol/liter) on basal cAMP (left panel) and IP3 release (right panel) by pheochromocytoma slices. cAMP production is not altered, and the adenylate cyclase inhibitor SQ-22536 (10-4 mol/liter) is ineffective. The PLC inhibitor U-73122 (10-5 mol/liter) abolishes the IP3 response to orexins. The mean ± SEM of six separate experiments (pheochromocytomas 5–10) are shown. *, P < 0.01 vs. the respective baseline value; A, P < 0.01 vs. the respective control value.

View larger version (42K): [in this window] [in a new window]   Figure 4. Effects of the adenylate cyclase inhibitor SQ-22536 (10-4 mol/liter), the PKA inhibitor H-89 (10-5 mol/liter), the PLC inhibitor U-73122 (10-5 mol/liter), and the PKC inhibitor calphostin C (10-5 mol/liter) on epinephrine (upper panel) and norepinephrine (lower panel) responses of pheochromocytoma slices to 10-8 mol/liter orexins. The mean ± SEM of six separate experiments (pheochromocytomas 5–10) are shown. *, P < 0.01 vs. the respective baseline value; A, P < 0.01 vs. the respective control value.

Our study shows that 10 benign secreting pheochromocytomas express OX2-R mRNA and possess a sizeable secretory response to both orexins A and B. These observations are in keeping with the fact that OX1-R binds orexin A, whereas OX2-R is nonselective for both orexins (1, 7).

It has been previously demonstrated that human adrenal medulla, although expressing high levels of OX1-R mRNA and low levels of OX2-R mRNA, does not respond to orexins (9). This finding may indicate that these receptors either possess a deficit in their coupling to G proteins and are silent receptors or mediate orexin functions other than stimulation of catecholamine secretion. Our present results make it likely that the neoplastic transformation of human adrenomedullary chromaffin cells associated with the repression of the OX1-R gene and the development of a rich population of well functioning OX2-R, the subtype of orexin receptor mainly expressed in the nervous system (17).

Our investigation strongly suggests that OX2-R located in pheochromocytoma cells act through the PLC-PKC signaling pathway. This view is supported by the following pieces of evidence. 1) Orexins do not enhance basal cAMP production by pheochromocytoma slices, but strongly increase IP3 release, an effect blocked by the PLC inhibitor U-73122 (18). 2) The catecholamine response to orexins is blocked by both U-73122 and the PKC inhibitor calphostin C (19) and is unaffected by either the adenylate cyclase inhibitor SQ-22536 (20) or the PKA inhibitor H-89 (21) at concentrations that were previously found to blunt the orexin-elicited glucocorticoid response of dispersed human and rat adrenocortical cells (8, 9). 3) Neither U-73122 nor calphostin C per se alters basal catecholamine secretion, which makes it unlikely that their effects ensue from a nonspecific cytotoxic action. This view is in keeping with the previous demonstration that in primary cultures of hypothalamic neurons orexins do not evoke any cAMP release, but stimulate PLC (2), OX2-R being coupled to G_i as well as G_q proteins (4). Theoretically, the interaction of OX2-R with G_i protein might also inhibit adenylate cyclase, a phenomenon undetectable under our experimental conditions. In this connection, it should be recalled that orexins A and B were found to decrease either pituitary adenylate cyclase-activating polypeptide-induced cAMP production or basal and pituitary adenylate cyclase-activating polypeptide-stimulated catecholamine synthesis in cultured rat pheochromocytoma PC12 cells (11). However, these cells, although possessing orexin-binding sites, do not express OX1-R and OX2-R mRNAs, thereby suggesting that they are provided with orexin receptors other than the classic ones (11).

Although the great variability of pheochromocytomas is well recognized (12), our study provides strong evidence that these tumors possess OX2-R positively coupled to catecholamine secretion through the PLC-PKC-dependent cascade. The pathophysiological relevance of these findings remains to be assessed, because it is not known whether orexins circulate in the periphery at a concentration sufficient to produce the herein-described secretagogue effects. However, it has been recently found that rat adrenal medulla expresses not only the OX1-R and OX2-R, but also the prepro-orexin gene (10). Hence, the possibility that orexins may control pheochromocytoma functions through a paracrine-autocrine mechanism merits further investigation.

Acknowledgments

Footnotes

Abbreviations: OX1-R, orexin receptor type 1; OX2-R, orexin receptor type 2.

Received February 2, 2001.

Accepted July 1, 2001.

References

1. Sakurai T, Ameniya A, Ishii M, et al. 1998 Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behaviour. Cell 92:573–585[CrossRef][Medline]
2. Van den Pol AN, Gao XB, Obrietan K, Kilduff TS, Belousov AB 1998 Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin. J Neurosci 18:7962–7971[Abstract/Free Full Text]
3. Wolf G 1998 Orexins: a newly discovered family of hypothalamic regulators of food intake. Nutrit Rev 56:172–189
4. Sakurai T 1999 Orexin and orexin receptors: implication in feeding behaviour. Regul Pept 85:25–30[CrossRef][Medline]
5. Chemelli R, Willie J, Sinton C, et al. 1999 Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98:437–451[CrossRef][Medline]
6. Lin L, Faraco J, Li R, et al. 1999 The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98:365–376[CrossRef][Medline]
7. De Lecea L, Kilduff TS, Peyron C, et al. 1998 The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci USA 95:322–327[Abstract/Free Full Text]
8. Malendowicz LK, Tortorella C, Nussdorfer GG 1999 Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade. J Steroid Biochem Mol Biol 70:185–188[CrossRef][Medline]
9. Mazzocchi G, Malendowicz LK, Gottardo L, Aragona F, Nussdorfer GG 2001 Orexin A stimulates cortisol secretion from human adrenocortical cells through activation of the adenylate cyclase-dependent signaling cascade. J Clin Endocrinol Metab 86:778–782[Abstract/Free Full Text]
10. López M, Señaris R, Gallego R, et al. 1999 Orexin receptors are expressed in the adrenal medulla of the rat. Endocrinology 140:5991–5994[Abstract/Free Full Text]
11. Nanmoku T, Isobe K, Sakurai T, et al. 2000 Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells. Biochem Biophys Res Commun 274:310–315
12. Keiser HR 1995 Pheochromocytoma and related tumors. In: De Groot LJ, ed. Endocrinology. Philadelphia: Saunders; 1853–1877
13. Nussdorfer GG 1996 Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol Rev 48:495–530[Medline]
14. Mazzocchi G, Rossi GP, Neri G, Malendowicz LK, Albertin G, Nussdorfer GG 1998 11ß-Hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex. FASEB J 12:1533–1539[Abstract/Free Full Text]
15. Tso JY, Sun XH, Kao TH, Reece KS, Wu R 1985 Isolation and characterization of rat and human glyceraldehyde-3-phosphate cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13:2485–2502[Abstract/Free Full Text]
16. Neri G, Andreis PG, Prayer-Galetti T, Rossi GP, Malendowicz LK, Nussdorfer GG 1996 Pituitary adenylate cyclase-activating peptide (PACAP) enhances aldosterone secretion of human adrenal gland: evidence for an indirect mechanism probably involving the local release of catecholamines. J Clin Endocrinol Metab 81:169–173[Abstract]
17. Trivedi P, Yu H, Mac Neil DJ, van der Ploeg LHT, Guan XM 1998 Distribution of orexin receptor mRNA in the rat brain. FEBS Lett 438:71–75[CrossRef][Medline]
18. Thompson AK, Mostafapou SP, Denliger LC, Bleadale JE, Fischer SK 1991 The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. J Biol Chem 266:23856–23862[Abstract/Free Full Text]
19. Kobayashi E, Ando K, Nakano H, Iida T, Ohno H, Morimoto M, Tamaoki T 1989 Calphostins (UCN-1028), novel and specific inhibitors of protein kinase C. I. Fermentation, isolation, physico-chemical properties and biological activities. J Antibiot 42:1470–1474[Medline]
20. Goldsmith BA, Abrams TW 1991 Reversal of synaptic depression by serotonin at Aplysia sensory neuron synapses involves activation of adenyl cyclase. Proc Natl Acad Sci USA 88:9021–9025[Abstract/Free Full Text]
21. Chijiwa T, Mishima A, Hagiwara M, et al. 1990 Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272[Abstract/Free Full Text]

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mazzocchi, G. Articles by Nussdorfer, G. G. Search for Related Content PubMed PubMed Citation Articles by Mazzocchi, G. Articles by Nussdorfer, G. G. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Adrenal Gland Cancer Pheochromocytoma