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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).
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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).
|
). The potency
(EC50) and efficacy (percent increase elicited by
the maximal effective concentration) of orexins A and B were not
significantly different (Table 2).
|
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). Neither 10-4
mol/liter SQ-22536 nor 10-5 mol/liter H-89
affected the catecholamine response to orexins
(10-8 mol/liter), which conversely was abolished
by both 10-5 mol/liter U-73122 and
10-5 mol/liter calphostin C (Fig. 4).
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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 Gi as well as Gq proteins (4). Theoretically, the interaction of OX2-R with Gi 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
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