Elsevier

Neuropharmacology

Volume 71, August 2013, Pages 27-36
Neuropharmacology

Central functional response to the novel peptide cannabinoid, hemopressin

https://doi.org/10.1016/j.neuropharm.2013.03.007Get rights and content

Highlights

  • Hemopressin is the first peptide ligand for the cannabinoid 1 (CB1) receptor.

  • Hemopressin selectively modulates nociception and appetite.

  • We resolved the neuronal circuitry mediating hemopressin's actions.

  • Hemopressin modulates nuclei known to modulate appetite and nociception.

  • Hemopressin does not activate reward brain centres.

Abstract

Hemopressin is the first peptide ligand to be described for the CB1 cannabinoid receptor. Hemopressin acts as an inverse agonist in vivo and can cross the blood–brain barrier to both inhibit appetite and induce antinociception. Despite being highly effective, synthetic CB1 inverse agonists are limited therapeutically due to unwanted, over dampening of central reward pathways. However, hemopressin appears to have its effect on appetite by affecting satiety rather than reward, suggesting an alternative mode of action which might avoid adverse side effects. Here, to resolve the neuronal circuitry mediating hemopressin's actions, we have combined blood-oxygen-level-dependent, pharmacological-challenge magnetic resonance imaging with c-Fos functional activity mapping to compare brain regions responsive to systemic administration of hemopressin and the synthetic CB1 inverse agonist, AM251. Using these complementary methods, we demonstrate that hemopressin activates distinct neuronal substrates within the brain, focused mainly on the feeding-related circuits of the mediobasal hypothalamus and in nociceptive regions of the periaqueductal grey (PAG) and dorsal raphe (DR). In contrast to AM251, there is a distinct lack of activation of the brain reward centres, such as the ventral tegmental area, nucleus accumbens and orbitofrontal cortex, which normally form a functional activity signature for the central action of synthetic CB1 receptor inverse agonists. Thus, hemopressin modulates the function of key feeding-related brain nuclei of the mediobasal hypothalamus, and descending pain pathways of the PAG and DR, and not higher limbic structures. Thus, hemopressin may offer behaviourally selective effects on nociception and appetite, without engaging reward pathways.

Introduction

To date, all known endogenous cannabinoids, such as 2-arachidonoylglycerol (2-AG) and anandamide, are eicosanoid fatty acid derivatives (Bisogno, 2008; Petrosino and Di Marzo, 2010). These endocannabinoids are released by postsynaptic neurons “on demand,” following the Ca2+ influx produced in response to postsynaptic depolarization or activation of metabotropic receptors (Kano et al., 2009). When released into the synaptic cleft, endocannabinoids activate presynaptic cannabinoid 1 (CB1) receptors, and impart an inhibitory action on further presynaptic transmission (Di Marzo et al., 2004; Wilson and Nicoll, 2002). Pharmacological manipulation, coupled with expression studies, has implied integral roles for CB1 in a diverse range of physiological functions, including memory, cognition, reward, appetite and nociception (Pertwee, 2005; Valverde et al., 2005). Due to this diverse functionality, CB1 receptors have immense therapeutic potential and are, in fact, established targets particularly in nociceptive and metabolic disorders (Di Marzo, 2011; Di Marzo et al., 2011; Walker and Hohmann, 2005).

The development of CB1-based therapies, particularly as anti-obesity treatments, has been hindered following the withdrawal of rimonabant (Acomplia; SR141716) by the European Medicines Agency. Rimonabant is a synthetic CB1 inverse agonist, capable of producing weight-reducing effects over extended periods in rodents and humans (Di Marzo, 2008; Van Gaal et al., 2005). Rimonabant antagonises central CB1 receptors to acutely reduce motivational appetite (Griebel et al., 2005; Thornton-Jones et al., 2005, 2007). This effect involves a corticostriatal–hypothalamic pathway thought to provide a link whereby reward/motivational circuits impinge on the hypothalamic control of feeding (Dodd et al., 2009; Kelley et al., 2005). In addition to affecting motivational appetite, rimonabant reduces body-fat mass via peripheral interaction with lipid mobilization pathways in adipose tissue and liver, and via energy expenditure and cellular glucose uptake (Di Marzo, 2008; Dodd et al., 2009; Kunos et al., 2009; Nogueiras et al., 2008). Despite being an effective anti-obesity treatment, the withdrawal of rimonabant was a consequence of undesirable central nervous system effects resulting in depression, anxiety or suicidality (Christensen et al., 2007). The discovery of selective CB1 antagonists capable of maintaining therapeutic benefits whilst lacking these adverse side effects is highly coveted.

Hemopressin is a nine-amino acid peptide, derived from the alpha chain of haemoglobin, which was discovered originally using substrate capture on rat brain homogenates (Heimann et al., 2007; Rioli et al., 2003). Hemopressin acts in vitro to functionally antagonise CB1 receptors, and demonstrates selective CB1 inverse agonism with sub-nanomolar potency (Dodd et al., 2010b; Heimann et al., 2007). Like rimonabant, hemopressin inhibits food intake in both normal and obese rodent models, and hemopressin can block CB1 agonist-induced hyperphagia in vivo (Dodd et al., 2010b). Interestingly, whereas rimonabant's anorectic effects are by inhibiting hedonic motivation, hemopressin appears to have a specific effect on satiety rather than on reward, and does not exhibit some of rimonabant's off-target behavioural side effects, at least in rodents (Dodd et al., 2009, 2010b; Kirkham, 2009). Hemopressin also induces robust non-opioid antinociception (Heimann et al., 2007), again devoid of non-selective behavioural effects, such as hypothermia, catalepsy and hypoactivity, seen previously with synthetic CB1 ligands (Hama and Sagen, 2011b; Rahn and Hohmann, 2009). Taken together, these studies suggest that hemopressin may confer its anorectic and antinociceptive actions via more defined CB1-mediated mechanisms. However, owing to its small size and high sequence homology with haemoglobin, hemopressin's expression, mode of action, and the neuronal targets underlying the peptide's behavioural effects are yet to be explored fully. Understanding the central circuits mediating hemopressin's action at a whole-brain level may be of high significance as it will offer a deeper insight into modes of CB1 receptor antagonism.

By using the complementary techniques of c-Fos immunohistochemical activity mapping and blood-oxygen-level-dependent (BOLD), pharmacological-challenge magnetic resonance imaging (phMRI), we aim to describe the central circuitry modulated by hemopressin in rodents. Through comparison with the effects in CB1−/− mice and to that of the rimonabant structural analogue, AM251, we hypothesise that hemopressin may act as a functional antagonist at CB1 receptors capable of selectively modulating the activity of hypothalamic appetite centres in the brain.

Section snippets

Animals

Experiments were carried out using adult male Sprague–Dawley rats, CD1 mice (Charles River Laboratories, Inc., Sandwich, UK) and CB1+/+ and CB1−/− littermate mice (Marsicano et al., 2002). Animals were group housed in The University of Manchester animal unit in a constant environment of 21 ± 2 °C and 45 ± 10% humidity, on a 12:12 h light–dark cycle with the dark phase commencing at 20:00. Chow (Beekay International, Hull, UK) and tap water were available ad libitum. All procedures conformed

Effects of hemopressin and the synthetic CB1 inverse agonist, AM251, on c-Fos immunoreactivity

To demonstrate that hemopressin and AM251 were effective in these animals, food intake was significantly decreased 90 min after injection (vehicle: 1.3 ± 0.1 g, hemopressin: 0.8 ± 0.1 g, p < 0.05; vehicle: 1.2 ± 0.2 g, AM251: 0.4 ± 0.1 g, p < 0.05). Quantitative analysis of the number of c-Fos-positive neurones in the brain regions of interest revealed a significant increase in counts following hemopressin (Figs. 1 and 3) administration. Hemopressin, when compared with vehicle injection,

Discussion

Despite growing evidence suggesting that hemopressin plays a behaviourally selective role in both CB1-mediated nociception and appetite (Dodd et al., 2010a; Heimann et al., 2007), nothing is known of the underlying neuronal mechanisms. To address this at the ‘whole-brain’ level, we have employed complementary BOLD phMRI and c-Fos protein functional activity mapping to spatially resolve the underlying neuronal circuitry modulated by hemopressin and the synthetic CB1 inverse agonist, AM251. Our

Disclosure statement

The authors Garron T. Dodd, Amy A. Worth, Duncan J. Hodkinson, Raj K. Srivastava, Beat Lutz, Steven R. Williams, and Simon M. Luckman all declare no conflict of interest.

Acknowledgements

The authors wish to thank the technical assistance of Miss Katie Murray for assistance with femoral artery cannulations, and Ms Karen Davies for maintenance of the MRI magnet and console. This work was supported by a Biotechnology and Bioscience Research Council grant.

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