Orexin-A expression in human peripheral tissues
Introduction
Orexins (hypocretins) are neuropeptides which were originally discovered from the rat brain (De Lecea et al., 1998, Sakurai et al., 1998). Orexins consist of two peptides, orexin-A (a 33 amino acid peptide) and orexin-B (a 28 amino acid peptide), both of which are derived from the same precursor prepro-orexin by proteolytic processing (De Lecea et al., 1998, Sakurai et al., 1998). Results of previous studies have indicated that orexin-containing neurons are present in the lateral hypothalamic area of the brain, which is, in general, considered the feeding center (De Lecea et al., 1998, Sakurai et al., 1998). Nerve fibers contained orexin-A and/or orexin-B have also been demonstrated to project widely from the hypothalamus to various brain regions (Peyron et al., 1998, Date et al., 1999, Harrison et al., 1999, van den Pol, 1999). Orexins in these brain structures may be involved in regulating various brain functions such as feeding (De Lecea et al., 1998, Sakurai et al., 1998) and sleeping (Chemelli et al., 1999).
Recently, the presence of orexin-A has been reported in human plasma (Arihara et al., 2001, Dalal et al., 2001). Orexin-A in plasma has also been demonstrated in patients with narcolepsy (Dalal et al., 2001, Higuchi et al., 2002), in whom production of orexin-A has been shown to be markedly reduced in the hypothalamus (Nishino et al., 2000, Peyron et al., 2000, Thannickal et al., 2000). These findings in humans may suggest the possible presence of orexin-A production in peripheral tissues outside the brain (Dalal et al., 2001, Higuchi et al., 2002). Previously, Kirchgessner and Liu (1999) reported the expression of orexin-A in the gut and pancreas. However, little is known about the expression and biological significance of orexin-A in systemic or peripheral human tissues. Therefore, in this study, we examined the systemic distribution of orexin-A in human tissues using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), as a first step towards understanding the biological significance of orexin-A in human peripheral tissues.
Section snippets
Antibody
The generation and characterization of the primary polyclonal antibody for orexin-A has been described previously (Arihara et al., 2000). Briefly, the antiserum against human orexin-A (No 677) was raised in a rabbit. Synthetic orexin-A (Peninsula Laboratories Inc., Belmont CA, USA) was conjugated to bovine serum albumin (Sigma Chemical Co., St Louis, MO, USA) by carbodiimide (Peptide Institute Inc., Minoh-shi, Japan), and injected into a rabbit with complete Freund adjuvant (Difco Laboratories,
Immunohistochemical localization of orexin-A in human tissues
In the positive control specimens, immunoreactivity for orexin-A was detected in the cytoplasm of neurons of the lateral hypothalamic area of the brain (Fig. 1A), as reported previously (De Lecea et al., 1998, Sakurai et al., 1998). No immunoreactivity was detected in sections of the lateral hypothalamic area used in the preabsorption test for orexin-A (Fig. 1B).
Results of immunohistochemical staining for orexin-A in human peripheral tissues are summarized in Table 1. Orexin-A immuno-positive
Discussion
In our present systemic study of human peripheral tissues, orexin-A immunoreactivity was detected in the ganglion cells of the thoracic sympathetic trunk and myenteric plexuses of the gastrointestinal tract, but was not detected in adrenal medullary chromaffin cells. These cells are considered to be derived from the same origin, the neural crest (Pomeranz and Gershon, 1990). In the process of embryonic development, the neural crest is known to distinctly differentiate into neuroblasts which
Acknowledgements
We thank Dr Yin Li (Department of Molecular Biology and Applied Physiology, Tohoku University) for technical assistance in the DNA sequencing.
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