Elsevier

Neuropharmacology

Volume 42, Issue 1, January 2002, Pages 127-133
Neuropharmacology

SB-334867-A antagonises orexin mediated excitation in the locus coeruleus

https://doi.org/10.1016/S0028-3908(01)00156-3Get rights and content

Abstract

Electrophysiological recordings from identified noradrenergic locus coeruleus (LC) neurones in rat brain slices have revealed that the orexins can cause direct and reversible depolarisation of the postsynaptic membrane. Whilst it is known that the membrane depolarisation produced by orexin-A can triple the firing rate of spontaneously active LC neurones, quantitative pharmacological analysis that determines the receptor subtype(s) mediating the orexinergic response has not yet been performed. Here we demonstrate that the effects of orexin-A are five-fold more potent than orexin-B on LC neuronal excitability. We show further that the orexin receptor antagonist SB-334867-A inhibits the effects of both agonists with pKB values similar to those calculated for human OX1 receptors expressed in CHO cells. Finally, we found no evidence for tonic activation of OX1 receptors in LC noradrenergic neurones despite electron microscopic evidence that orexin terminals directly contact these neurones. These data demonstrate that SB-334867-A is a useful tool compound with which to study the physiology of OX1 receptors.

Introduction

The orexins/hypocretins exist as two neuropeptides referred to as orexin-A (hypocretin 1) and orexin-B (hypocretin 2) which are derived from the precursor molecule prepro-orexin (De Lecea et al., 1998, Sakurai et al., 1998). Each exhibits different affinities for the two orexin receptors that have so far been identified. In particular, orexin-A has higher affinity than orexin-B for the OX1 receptor whereas both orexin-A and -B have equal affinities for the OX2 receptor (Smart et al., 1999, Smart et al., 2001). The native signal transduction pathways for these G protein coupled receptors have not yet been precisely defined but studies on the cloned receptors have demonstrated that activation of OX1 receptors causes a concentration-dependent mobilisation of calcium in CHO and HEK293 cells that is most likely mediated through coupling to the GQ type of G protein (Sakurai et al., 1998, Smart et al., 1999).

Irrespective of this, the discovery of these agonist molecules has stimulated investigation into the physiological roles that they fulfil. In particular, immunohistochemical and electron microscopic analysis has revealed extensive projections of hypothalamic afferents containing orexins to numerous brain areas where they make asymmetrical synaptic contacts with their target neurones (Peyron et al., 1998, Horvath et al., 1999). Functional studies using exogenous application of orexins have confirmed the presence of orexin receptors in many brain structures, including the locus coeruleus, LC (Hagan et al., 1999, Horvath et al., 1999, Ivanov and Aston-Jones, 2000), hypothalamus (Van den Pol et al., 1998, Date et al., 1999) and raphe nucleus (Brown et al., 2001), suggesting that the orexin receptor system is important in physiological functions such as control of sleep–wake cycles, nociception, arousal and feeding (Dube et al., 1998, Sakurai et al., 1998, Haynes et al., 2000, Bingham et al., 2001, Duxon et al., 2001, Jones et al., 2001).

As immunohistochemical and in situ hybridisation analysis has pointed towards differential expression of the OX1 and OX2 orexin receptor subtypes in distinct brain regions (Trivedi et al., 1998, Greco and Shiromani, 2001, Hervieu et al., 2001) it is possible that pharmacological agents tailored to interfere with either OX1 or OX2 receptors will selectively modify one or other of the physiological functions listed above. However, to date, quantitative pharmacological methods definitively proving the nature of the receptor mediating individual effects of orexins are scarce. Part of the problem has been the lack of selective antagonists for each orexin receptor subtype. Recently, the first selective orexin receptor antagonist, SB-334867-A, has been developed (Haynes et al., 2000, Rodgers et al., 2001, Smart et al., 2001). In recombinant expression systems SB-334867-A displays a 20-fold higher affinity for the OX1 receptor over the OX2 receptor (Smart et al., 2001). Potentially, this compound could prove useful in defining the physiological function of the OX1 receptor. However, G protein coupled receptors can exhibit different apparent affinity/efficacy states which are often exaggerated in recombinant systems due to the constrained configuration in which receptors have been expressed and the methods used to assess their function (Kenakin, 1999). As such, it is important to establish whether SB-334867-A acts as a competitive antagonist of orexin receptors in native tissue and, more importantly, whether it exhibits the same affinity to that which has been defined in recombinant systems. To achieve these goals, we have studied the effects of SB-334867-A in the LC, a region of the brain believed to express predominantly the OX1 receptor (Greco and Shiromani, 2001, Hervieu et al., 2001). Some of these results have been published previously in abstract form (Evans et al., 1999).

Section snippets

Electrophysiology

All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986. Coronal or horizontal slices (400 μm thick) through the LC of male Sprague-Dawley rats (4–8 weeks old) were cut (in a solution of composition, in mM: sucrose 120, KCl 2.5, CaCl2 0.1, MgCl2 5, NaHCO3 25, NaHPO4 2.5, glucose 10) at 2–6°C using a vibrating blade tissue slicer (Camden). Slices were left to equilibrate for at least one hour in artificial cerebrospinal fluid (ACSF, composition

Materials

Orexin-A, orexin-B, and SB-334867-A were obtained from the Department of Medicinal Chemistry, GlaxoSmithKline, Harlow. All other compounds were obtained from sources as follows: tetrodotoxin (TTX, Calbiochem), cadmium (Sigma), 3,5-dihydroxyphenylglycine (DHPG) and oxotremorine-M (Tocris). Synthetic orexin-A, orexin-B and 1-(2-methylbenzoxazol-6-yl)-3-[1,5]napthyridin-4-yl-urea hydrochloride (SB-334867-A) were dissolved in distilled water (orexins) or DMSO (SB-334867-A) to form stock solutions

Results

Previous reports have demonstrated that LC neurones are responsive to exogenous application of orexin-A (Hagan et al., 1999, Horvath et al., 1999, Ivanov and Aston-Jones, 2000). We have confirmed and extended these studies, and showed that orexin-A caused a concentration dependent increase in single unit firing in LC neurones. This increase was sustained for the period of agonist application, exhibiting little if any desensitisation (n=8; Fig. 1A). The potentiation of cell firing was reversible

Discussion

The data presented here are consistent with previous studies in demonstrating that orexin increases neuronal excitability in the LC through direct membrane depolarisation which leads to increased probability of action potential firing (Hagan et al., 1999, Horvath et al., 1999, Ivanov and Aston-Jones, 2000). In addition, we now show that this orexin-induced response does not desensitise or run down even in extracellular medium devoid of calcium, and, as such, repeated applications of orexin

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      Orexin receptors are differentially distributed on soma as well as both pre- and post-synaptic membranes of target cells throughout the central nervous system (CNS) and this may somehow explain the multifaceted physiological functions mediated by the activity of endogenous orexin system [5,8]. Considerable number of studies have demonstrated the functional expression of orexin receptors in various brain regions such as locus coeruleus (LC) [9–12], ventral tegmental are (VTA) [13–15], raphe nuclei [16–18], thalamic and hypothalamic structures [19–21], periaquiductal gray (PAG) [22–24], lateral paragigantocellularis (LPGi) [25–27], tuberomammillary nucleus (TMN) [28,29], nucleus accumbens (NAc) [30,31], olfactory system, septal nuclei, olivary complex, dorsal root ganglia (DRG) etc. [29]. It is noteworthy that [13]among these brain structures, LC has been shown to display the highest density of OX1R immunoreactivity [29].

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