Evidence for a modulatory role of orexin A on the nitrergic neurotransmission in the mouse gastric fundus Maria Caterina Baccari a, ⁎, Daniele Bani b , Franco Calamai a a Department of Physiological Sciences, University of Florence, Florence, Italy b Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy abstract article info Article history: Received 29 July 2008 Received in revised form 12 November 2008 Accepted 15 December 2008 Available online 31 December 2008 Keywords: Nitric oxide Neuromodulation Non-adrenergic Non-cholinergic The presence of orexins and their receptors in the gastrointestinal tract supports a local action of these peptides. Aim of the present study was to investigate the effects of orexin A (OXA) on the relaxant responses of the mouse gastric fundus. Mechanical responses of gastric strips were recorded via force-displacement transducers. The presence of orexin receptors (OX-1R) was also evaluated by immunocytochemistry. In carbachol precontracted strips and in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. All relaxant responses were abolished by TTX. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-NNA (2 × 10 -4 M) as well as by the guanylate cyclase inhibitor ODQ (1×10 -6 M). OXA (3×10 -7 M) greatly increased the amplitude of the EFS-induced fast relaxation without affecting the sustained one. OXA also potentiated the amplitude of the relaxant responses elicited by the ganglionic stimulating agent DMPP (1 ×10 -5 M), but had no effects on the direct smooth muscle relaxant responses elicited by papaverine (1 × 10 -5 M) or VIP (1 × 10 -7 M). In the presence of L-NNA, the response to DMPP was reduced in amplitude and no longer influenced by OXA. The OX1 receptor antagonist SB-334867 (1 × 10 -5 M) reduced the amplitude of the EFS-induced fast relaxation without influencing neither the sustained responses nor those to papaverine and VIP. Immunocytochemistry showed the presence of neurons that co-express neuronal nitric oxide synthase and OX-1R. These results indicate that, in mouse gastric fundus, OXA exerts a modulatory action at the postganglionic level on the nitrergic neurotransmission. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Orexins (orexin A and orexin B) are a family of hypothalamic neuropeptides that appear to exert important effects on arousal and sleep-wake cycle [1–3], as well as on appetite and regulation of feeding and energy homeostasis [2,4]. Both orexin-A (OXA) and orexin-B (OXB) act through two subtypes of receptors, named OX-1R and OX-2R, that belong to the G protein-coupled superfamily of receptors [5–7]. The OX-1R almost exclusively binds orexin-A while OX-2R is non-selective and binds both orexins. Even if both peptides have been reported to increase food intake, OXA appears to be more effective in stimulating feeding, drinking and gastric secretion than OXB [8–12], thus suggesting that OXA is closely related with the regulation of food intake and gastric functions. Orexins and orexins receptors are present not only in the central nervous system but also in peripheral tissues, such as the gastro- intestinal tract, where they have been located in the enteric nervous system as well as at the smooth muscle level [13–15]. The abundant distribution of orexins and their receptors in the gut supports the local influence of these peptides in several functions, including motility: in fact, both contractile and relaxant effects of OXA have been described in the gastrointestinal tract [14]. Interestingly, in some enteric neurons, orexin immunoreactivity shares localization with inhibitory non-adrenergic, non-cholinergic (NANC) neurotransmitters, including nitric oxide (NO) which is considered as the main inhibitory substance for gastrointestinal relaxation [16–18]. A subset of OX1-R-immunoreactive neurons has been reported to contain neuronal nitric oxide synthase (nNOS) in the myenteric plexus of the rat small bowel [19]. Furthermore, in the rat distal stomach, OXA-positive nerve fibers were found in close proximity to nNOS immunoreactive cell bodies of the myenteric plexus and OXA-immunoreactive nerve fibers co-express nNOS in ganglia as well as in circular muscle [20]. Besides the immunohisto- chemical evidence, a close link between OXA and NO is also supported by functional studies: in the small intestine of rodents, this peptide has been suggested to mediate NANC relaxation through activation of nitrergic neurons and to inhibit fasting motility via the L-arginine-NO pathway [15,19,21]. Recently, in fundal strips from mice, OXA has been observed to depress the amplitude of the neurally-induced contractile responses, an effect antagonized by the NO synthesis inhibitor L-NNA Regulatory Peptides 154 (2009) 54–59 ⁎ Corresponding author. Department of Physiological Sciences, University of Florence, Viale G.B. Morgagni 63, I-50134, Florence, Italy. Tel.: +39 55 4237311; fax: +39 55 4379506. E-mail address: mcaterina.baccari@unifi.it (M.C. Baccari). 0167-0115/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2008.12.005 Contents lists available at ScienceDirect Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep