Elsevier

Neuropharmacology

Volume 89, February 2015, Pages 412-423
Neuropharmacology

HINT1 protein: A new therapeutic target to enhance opioid antinociception and block mechanical allodynia

https://doi.org/10.1016/j.neuropharm.2014.10.022Get rights and content

Highlights

  • The HINT1 protein engages NMDAR negative control on MOR signaling.

  • The HINT1 enzymatic inhibitor TpGc disconnects MOR effects from NMDAR control.

  • TpGc enhances morphine antinociception and reduces acute tolerance.

  • TpGc reduces allodynia in the CCI animal model of neuropathic pain.

  • A single icv injection of 20 nmol TpGc alleviates CCI-evoked allodynia for three days.

Abstract

In the nervous system, the glutamate N-methyl-d-aspartate receptor (NMDAR) restricts the activity of the mu-opioid receptor (MOR). Both receptors are present in midbrain periaqueductal grey (PAG) neurons, an area that plays a central role in the supraspinal antinociceptive effects of opioids. The cross-talk that occurs between these receptors is sustained by the MOR-associated histidine triad nucleotide binding protein 1 (HINT1), which displays nucleoside phosphoramidase and acyl-AMP hydrolase activity. Here we report that the inhibitor of HINT1 enzymatic activity guanosine-5′-tryptamine carbamate (TpGc) significantly enhanced morphine antinociception while preventing the development of tolerance. At the molecular level, TpGc reduced the capacity of MORs to recruit NMDAR activity to negatively regulate opioid signaling. In mice suffering from chronic constriction injury concurrent with increased NMDAR activity, a single intracerebroventricular administration of TpGc attenuated NMDAR function and alleviated mechanical allodynia for several days. These data suggest a potential therapeutic role for HINT1 inhibitors in the clinical management of acute and neuropathic pain.

Introduction

The Mu-opioid receptor (MOR1) is a G-protein-coupled receptor (GPCR) that selectively controls the perception of nociceptive sensory signals and, the activation of the MOR by morphine and its derivatives produces high levels of antinociception. The frequent administration of exogenous opioids typically leads to the development of antinociceptive tolerance as a consequence of MOR desensitization. In nervous tissue, the N-methyl-d-aspartate acid glutamate receptor (NMDAR2)/neural nitric oxide synthase (nNOS)/calcium (Ca2+) and calmodulin (CaM)-dependent kinase II (CaMKII) pathway plays an essential role in MOR desensitization (Pasternak et al., 1995, Trujillo, 2002). The MOR interacts with the NR13 subunit of the NMDAR (Rodríguez-Muñoz et al., 2012) and positively regulates NMDAR calcium fluxes (Chen and Huang, 1991) that, via the activation of calmodulin, negatively regulate the signaling strength of the MOR (Garzón et al., 2012, Rodríguez-Muñoz and Garzón, 2013). The MOR-NMDAR association requires histidine triad nucleotide-binding protein 1 (HINT1). In the absence of HINT1 this physical interaction weakens, and morphine does not stimulate NMDAR activity (Rodríguez-Muñoz et al., 2011a).

The HINT1 protein is widely expressed in the CNS (Liu et al., 2008), where it interacts with the C terminal cytosolic sequence of the MOR and with the C1 cytosolic segment of the NMDAR NR1 subunit (Guang et al., 2004, Sánchez-Blázquez et al., 2013b). Thus, HINT1 behaves as a scaffold protein that sustains the regulatory crosstalk of MORs with NMDARs (Rodríguez-Muñoz et al., 2012). Consequently, in HINT1-deficient mice, NMDAR fails to exert any negative control over MOR signaling. Therefore, HINT1−/− mice exhibit enhanced morphine-induced antinociception, which eventually provokes an antinociceptive tolerance that is heterologous and independent of NMDAR activity (Rodríguez-Muñoz et al., 2011a).

The HINT1 protein was initially known as protein kinase C-interacting protein (PKCi) because it binds to and inhibits the function of PKC. HINT1 is a highly conserved 14 KDa protein that belongs to the histidine triad superfamily (HIT), the members of which contain the HisXHisXHisXX motif (where X is a hydrophobic amino acid) for which HIT proteins are named (Brenner, 2002). HINT1 exhibits nucleoside phosphoramidase and acyl-AMP hydrolase activity (Brenner, 2002, Chou et al., 2005, Chou et al., 2007a, Chou et al., 2007b). HINT1 prefers purine over pyrimidine phosphoramidates, and the 2- and 3-hydroxyl groups of the ribose ring are required for optimal phosphoramidase efficiency (Chou et al., 2007a). In addition, the catalytic mechanism of HINT1 for either nucleoside phosphoramidate or acyl-AMP substrates has been found to proceed through rapid formation of nucleotidylated His-112, followed by partially rate limiting hydrolysis and release of nucleotide monophosphate (Zhou et al., 2013).

HINT1 exists as a zinc-independent homodimer in which the two protomers associate through their C-terminal sequences to form a negatively charged surface (Lima et al., 1996). The two sets of histidines 110, 112 and 114 (plus histidine 51) near the carboxyl terminal end are exposed to interact in a zinc-dependent manner with third-party proteins (Pearson et al., 1990, Rodríguez-Muñoz et al., 2011a). In humans, three HINT proteins have been described: HINT1, HINT2 and HINT3. In mice and at the level of the neuronal membrane, HINT1 plays a relevant role in the regulation of MORs and potentially other GPCRs as well (Ajit et al., 2007, Guang et al., 2004, Rodríguez-Muñoz et al., 2008). Thus, gene association studies have linked nicotine dependence with Hint1 gene variants (Jackson et al., 2013), and HINT1-deficient mice present enhanced locomotion in response to amphetamine and the dopamine receptor agonist apomorphine (Barbier et al., 2007). Hence, a lack of HINT1 may also be associated with the deregulation of postsynaptic dopamine transmission (Barbier et al., 2007). Moreover, mutated forms of the HINT1 protein have been associated with neurodegenerative disorders such as axonal neuropathy with neuromyotonia (Zimon et al., 2012).

Thus, HINT1 behaves as a scaffold protein at the cell membrane, the activity of which is regulated by redox processes and most likely also by its binding to ribonucleosides (Garzón et al., 2012, Vicente-Sánchez et al., 2013). Histidine triad nucleotide binding proteins represent the most ancient branch of the histidine triad (HIT) protein superfamily, and are found in all branches of the tree of life. Catalytically active Escherichia coli HinT was found to be essential for the activity of the d-alanine dehydrogenase enzyme (Bardaweel et al., 2011), a finding that led to the development of cell-permeable HINT1 inhibitors (Bardaweel et al., 2012). These tools open up the possibility of studying processes that are potentially regulated by HINT1 at the cell membrane, such as MOR-NMDAR cross-regulation. Therefore, we addressed the possible influence of the HINT enzymatic inhibitor guanosine-5′-tryptamine carbamate (TpGc) (Bardaweel et al., 2011, Bardaweel et al., 2012) on the capacity of morphine to promote antinociception and in the possible development of antinociceptive tolerance. The intracerebroventricular (icv) administration of TpGc to mice potentiated morphine-evoked antinociception and reduced the development of acute tolerance. Interestingly, a single icv administration of TpGc to mice suffering chronic constriction injury (CCI) led to a significant attenuation of mechanical allodynia that persisted for several days, suggesting a regulatory role for HINT1 in its interaction with NMDARs.

Section snippets

Animals and intracerebroventricular injection

Male albino CD-1 mice (Charles River), and a mouse knock-out strain on a 96% 129 mice genetic background carrying a disrupted HINT1 allele and the corresponding wild type (a gift from I.B. Weinstein/J.B. Wang) (Su et al., 2003), were used in these studies. Genotypes were confirmed by PCR analysis of DNA extracted from tail biopsies, and the animals used in this study were 8- to 12-week-old adult male mice. The mice were maintained at 22 °C on a diurnal 12 h light/dark cycle. Procedures

HINT1 substrates and inhibitors

To assess the role of the HINT1 active site on nociception, we prepared an HINT substrate, adenosine 5′-N-tryptamine phosphoramidate (TpAd) (kcat = 2.1 s−1, Km = 0.13 μM, kcat/Km = 1.5 × 107 s−1/μM and the HINT inhibitor TpGc (Ki = 34 μM) (Bardaweel et al., 2012, Chou et al., 2007a). In addition, since we have previously demonstrated that binding to the HINT1 active site is dependent on the 2′- and 3′- hydroxyl groups, we prepared TpGcKp, the 2′-3′- isopropylidene protected analog of TpGc, as an

Discussion

The NMDAR regulates essential processes in the nervous system, such as synaptic plasticity, learning, memory formation, and cognition, and its activation results in the permeation of Ca2+ ions, a function that is enhanced or restricted by certain GPCRs (Lu et al., 1999, Salter and Kalia, 2004) under the control of the HINT1 protein (Rodríguez-Muñoz et al., 2011a, Sánchez-Blázquez et al., 2012). Thus, GPCRs such as the MOR positively regulate NMDAR-mediated calcium fluxes (Chen and Huang, 1991,

Acknowledgments

We would like to thank Gabriela de Alba and Carmelo Aguado for their excellent technical assistance. This research was supported by MSC ‘Plan de Drogas 2011–14’ and the Ministerio de Economía y Competividad (MINECO), SAF 2012-34991. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no conflicts of interest. CRW also wish to thank the University of Minnesota Foundation for partial support of this

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