Towards a Receptor for Nocistatin?

Abstract

Nocistatin is a peptide derived from the pronociceptin precursor, the source of nociceptin, the endogenous ligand for the nociceptin (NOP or ORL1). Despite nocistatin showing activity in a wide range of assays for nociception and other CNS activities, there is a dearth of information regarding the cellular actions of this peptide in the brain, and no receptor for nocistatin has been identified. In a study published in this issue of the British Journal of Pharmacology, Fantin and colleagues demonstrate that nocistatin inhibits 5-HT release from cortical synaptosomes in a concentration-dependent and Pertussis toxin-sensitive manner. The actions of nocistatin are independent of activity at NOP receptors. This study represents the first unambiguous demonstration of nocistatin agonist actions in brain and, taken together with previous work in the spinal cord, provides strong evidence that there is an as yet unidentified G protein-coupled receptor for nocistatin.

Neuropeptide transmitter systems are fascinating for many reasons, not the least of which is that neuropeptides are usually encoded by a larger precursor that contains several active peptides. Precursors can contain structurally distinct ligands for related receptors, such as the various opioid receptor ligands found in the proenkephalin and prodynorphin molecules while other precursors consist of ligands for very different receptors, such as the opioid agonist β-endorphin and multiple melanocortin receptor agonists found in proopiomelanocortin. Nociceptin, the endogenous ligand for the opioid-related receptor (NOP), is also derived from a larger precursor that encodes several other peptides with central nervous system activity. The best characterized of these peptides is nocistatin, so named because it blocked the pro-nociceptive effects of nociceptin when the peptides were injected together into the spinal cord of mice (Okuda-Ashitaka et al., 1998). The original study describing the actions of nocistatin showed that it had no activity at recombinant NOP receptors and also reported the specific binding of a nocistatin analogue to brain membranes. The minimum active sequence of nocistatin was soon confirmed across several species and nocistatin was identified in human brain (Lee et al., 1999). Identification of a receptor for nocistatin seemed only a matter of time.

We are still waiting.

Despite several dozen studies describing diverse biological actions of nocistatin, only small steps have been taken towards understanding the nature of its receptor. In the present issue of this Journal, Fantin and colleagues (Fantin et al., 2007) report another such step and, while it brings us no closer to the molecular identification of a nocistatin receptor, their results provide more strong evidence that a G protein-coupled receptor for nocistatin exists on neurons. In this study, Fantin etal. (2007) examined the effects of nocistatin on depolarization-evoked release of [³H]5-HT from cortical synaptosomes. The most important finding of the study is simply that nocistatin inhibited the release of [³H]5-HT in a concentration-dependent manner. They further showed that the effects of nocistatin did not depend on the expression or activity of NOP and they were blocked by loading the synaptosomes with Pertussis toxin, a selective inhibitor of signalling through G_i and G_o G proteins. To place these results into context, this study is the first to describe agonist actions of nocistatin in a system as simple as synapses or synaptosomes derived from brain. In an earlier series of landmark studies, Uli Zeilhofer and colleagues showed that nocistatin inhibited synaptic glycine and GABA release onto neurons in spinal cord slices and, as in the present study, they showed that the effects of nocistatin were abolished by Pertussis toxin and did not involve NOP receptors (Zeilhofer et al., 2000; Ahmadi et al., 2001, 2003).

The evidence for a nocistatin receptor seems compelling, so why do not we know more about it? A major problem has been the lack of tools to use to study the nocistatin-binding sites. The original radioligand used in the study of Okuda-Ashitaka et al. (1998) was not used again, and only very recently has an alternative strategy of using biotinylated nocistatin to label binding sites been reported (Kazi et al., 2007). It may be that the nocistatin receptor has quite a low abundance and a study which reported the anatomical location of nociceptin-stimulated G-protein activation using GTPγS labelling failed to find any evidence for a similar signal from a nocistatin receptor (Neal et al., 2003). The few studies that have successfully demonstrated agonist actions of nocistatin have studied neurotransmitter release, which may imply a selective localization of the receptor in presynaptic elements, which are hard to access directly. Finally, it is possible that nocistatin does act at an already identified receptor, but does so in an unusual manner that is not readily detected in screening assays, for example by acting as an allosteric enhancer of agonist action.

Research into the effects of nocistatin has been largely defined by its relationship with nociceptin and this may also have hampered progress towards its emergence as a neurotransmitter in its own right. Many studies with nocistatin in vivo have primarily investigated how it affects nociceptin actions, including whether it blocks the inhibitory effects nociceptin has on μ-opioid analgesia, and in vitro studies have often focussed on whether nocistatin mimicked or blocked the effects of nociceptin, despite there being very limited evidence that the two peptides interact at a cellular level (Nicol et al., 1998). The study of Fantin etal. (2007) offers an intriguing hint that there may be interactions between the signalling pathways utilized by the nocistatin receptor and NOP receptors because pretreatment with a high concentration of nocistatin partially attenuated the inhibition of [³H]5-HT release by a subsequent superfusion of nociceptin. In this experiment, the high concentration of nocistatin was by itself ineffective at inhibiting [³H]5-HT release, perhaps because of receptor desensitization. The effect on the nociceptin response suggests that this desensitization is at least partly heterologous.

We hope this study will revive interest in nocistatin signalling in the brain and stimulate a renewed effort to find the nocistatin receptor. Perhaps 5HT neurons in the raphe complex are a good place to begin to look, as it was in these neurons that the first neuronal actions of nociceptin were described (Vaughan and Christie, 1996). Nocistatin has been shown to affect a number of complex behaviours, most notably those reflecting the processing of noxious stimuli. The possibility that drugs acting at a nocistatin receptor might be analgesic is just one good reason to find a receptor for this orphan agonist.

Abbreviations

NOP

nociceptin receptor