Cellular localization of orexin receptors in human adrenal gland, adrenocortical adenomas and pheochromocytomas
Introduction
Orexins are hypothalamic peptides recently discovered and involved in the regulation of energy balance and arousal. They were identified by independent groups that named them hypocretin-1 and -2 [1] and orexin-A and -B [2], respectively. The sequences of hypocretin-1 and -2 overlapped with those of orexin-A and -B. However, hypocretins present additional amino acids and recent pharmacological studies demonstrated that hypocretins are weak agonists at orexin receptors [3]. Orexins arise by proteolysis from a 130-amino acid common precursor (prepro-orexin) [2]. Orexin-A and -B are 33 and 28 amino acids in length, respectively.
Orexins act on G protein previously orphan receptors named orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). OX1R is selective for orexin-A, whereas OX2R binds both orexin-A and -B [2]. OXR have a broad distribution in the brain [4] and it has demonstrated a synaptic action on NPY-producing neurones [5]. Orexins were first identified as neuropeptides that are expressed in the lateral hypothalamic area, a region of the brain classically implicated in feeding behaviour. However, the broad distribution of OXR in the brain suggests that they could have other functions apart from regulation of food intake [4]. In this sense, their involvement has been demonstrated in modulation of macrophage functions [6], control of the stress reaction, face washing, grooming and burrowing behaviours in rats [7], stimulation of gastric acid secretion [8] and regulation of the blood pressure, the neuroendocrine system, body temperature and sleep–waking cycle [9], [10], [11], [12], [13], [14]. Similarly, it has been demonstrated by double labelling that OX-positive nerve terminals are implicated in the control of sleep–wakefulness [15] and have been related with narcolepsy [16].
There is also evidence that orexins act on the pituitary–adrenocortical axis. It has been reported that the plasma concentrations of ACTH and corticosterone were increased after central administration of orexins [17] and that this influence is due to a central and peripheral action [18], [19]. Recently, we have demonstrated, by immunohistochemistry and RT-PCR, the expression of OXR in peripheral tissues, such as the rat adrenal gland [20] and the human pituitary [21]. The aim of the present work was to study the expression of OXR in normal human adrenal gland and to compare the OXR distribution in normal adrenal tissue and adrenal tumours (adrenocortical adenomas and pheochromocytomas).
Section snippets
Tissues
For this study, we have used normal human adrenal glands (n=7, three males and four females, 13–90 years old) obtained from surgical specimens or recent autopsies (<8 h postmortem), adrenocortical adenomas (n=4, one male and three females, 35–68 years old) and pheochromocytomas (n=8, four males and four females, 21–60 years old) from the Department of Pathology, University Clinical Hospital, Santiago de Compostela, Spain (Table 1).
Immunohistochemistry
Samples were immediately immersion-fixed in 10% buffered
Results
Intense immunoreactivity for OXR was demonstrated in normal human adrenal gland independent from sex and age. Immunopositivity for OX1R was found in the adrenal cortex without differences between the three layers (glomerulosa, fasciculata and reticularis) and no signal was observed in the medulla (Fig. 1A). Controls performed by preadsorption of the primary antibody with the homologous antigen (OX1R) were completely negative (Fig. 1B). Immunoreactivity for OX2R was restricted to the adrenal
Discussion
The present study describes for the first time the immunohistochemical expression of OXR in human adrenal gland. Immunoreactivity for OX1R was shown in glomerulosa, fasciculata, and reticularis zones of the adrenal cortex, whereas OX2R was found in virtually all cells of the adrenal medulla. Our results confirm the recent findings that human adrenocortical cells are mainly provided with OX1R [22]. In fact, these authors demonstrated by RT-PCR high levels of OX1R mRNA in human adrenal
Acknowledgements
We thank the expert technical assistance of D. Fernández-Roel and A. Vázquez Boquete. This work was supported by the Xunta de Galicia (grant no. PGIDT99PX120806B). M.B. is a recipient of a studentship from the Xunta de Galicia.
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