Binding and GTPγS autoradiographic analysis of preproorphanin precursor peptide products at the ORL1 and opioid receptors

Abstract

Utilizing agonist-stimulated GTPγS autoradiography, we analyzed the ability of preproorphanin FQ (ppOFQ) peptides to stimulate [³⁵S]-GTPγS binding in adult rat brain. Orphanin FQ (OFQ) stimulated [³⁵S]-GTPγS binding in a pattern similar to that described for [¹²⁵I]-OFQ at the endogenous opioid receptor-like (ORL1) receptor. The ppOFQ peptides nocistatin and orphanin FQ2 (OFQ II_1–17) had no effect, suggesting that they do not mediate their reported analgesic effects via a G_i/o-coupled receptor (i.e. opioid or ORL1). Unlike OFQ II_1–17, high concentrations of its C-terminal extension, OFQ II_1–28, stimulated [³⁵S]-GTPγS binding in a mu (μ) opioid receptor-like distribution and the effect was blocked by naloxone. To explore these observations, we evaluated the receptor binding profile of OFQ II_1–28 at the cloned ORL1 and μ opioid receptors. OFQ II_1–28 had no specific binding at either ORL1 or μ opioid receptors at concentrations up to 50 μM. This lack of affinity was not consistent with a μ-mediated effect, as suggested by preliminary observation using functional autoradiography in rat brain sections. Although behavioral studies suggest that OFQ II_1–28 possesses analgesic activity, this effect does not appear to be mediated via direct binding at the μ opioid receptor. Taken together, these findings support the view that (1) OFQ is the only ppOFQ peptide that binds to and activates the ORL1 receptor and (2) OFQ II_1–28 does not bind or stimulate [³⁵S]-GTPγS binding in cells expressing the μ opioid receptor.

Introduction

Binding of orphanin FQ (OFQ) to the opioid receptor-like (ORL1) receptor produces numerous functional effects. At the cellular level, activation of ORL1 inhibits cAMP accumulation, stimulates protein kinase C formation, and induces neuronal Ca²⁺ and K⁺ conductance changes. Systemic and central nervous system (CNS) infusions of OFQ have been shown to modulate several complex functions, including cardiovascular control, water balance, nociception, feeding, learning, locomotion, stress response, and sexual behavior (Civelli et al., 1998, Darland et al., 1998, Harrison and Grandy, 2000, Mogil and Pasternak, 2001).

The ORL1 receptor shares significant amino acid homology with the known opioid receptors (μ, κ, and δ). It contains seven transmembrane spanning domains and is a member of the G protein-coupled family of receptors (Bunzow et al., 1994, Chen et al., 1994, Marchese et al., 1994, Mollereau et al., 1994, Fukuda et al., 1994, Wick et al., 1994, Wang et al., 1994, Lachowicz et al., 1995). In spite of the structural similarities between ORL1 and endogenous opioid receptors, opioid peptides and alkaloids have little affinity for ORL1 (Bunzow et al., 1994, Chen et al., 1994, Fukuda et al., 1994, Lachowicz et al., 1995, Mollereau et al., 1994, Wick et al., 1994, Ma et al., 1997, Nicholson et al., 1998). OFQ, the endogenous ligand for ORL1, is strikingly similar to the opioid peptide dynorphin A_1–17 (Meunier et al., 1995, Reinscheid et al., 1995). However, in spite of this structural homology, OFQ has little binding affinity for the opioid receptors (Meunier et al., 1995, Reinscheid et al., 1996, Reinscheid et al., 1998).

Similar to the endogenous opioid peptides, OFQ is derived from a large precursor, preproorphanin FQ (ppOFQ; Fig. 1). This peptide is structurally similar to the opioid precursor prodynorphin (Mollereau et al., 1996, Houtani et al., 1996, Nothacker et al., 1996, Pan et al., 1996), and there is a high degree of ppOFQ conservation across human, bovine, rat, and mouse species (Nothacker et al., 1996, Civelli et al., 1998). It is believed that a coordinated mechanism of evolution separated the OFQ and opioid systems (Reinscheid et al., 1998, Danielson and Dores, 1999, Murphy et al., 2001). Similar to the opioid precursor molecules, recent studies indicate that ppOFQ contains other putative peptides that may be biologically active (Fig. 1) (Okuda-Ashitaka et al., 1998, Rossi et al., 1998, Hiramatsu and Inoue, 1999a, Hiramatsu and Inoue, 1999b, Xu et al., 1999, Yamamoto and Sakashita, 1999, Zhao et al., 1999, Nakano et al., 2000). Upstream from OFQ is a peptide sequence flanked by double basic amino acids that may be liberated with post-translational processing. This molecule, nocistatin (ppOFQ 98–132), appears to have significant analgesic activity and has been shown to antagonize OFQ nociceptive effects (Okuda-Ashitaka et al., 1998, Hiramatsu and Inoue, 1999b, Xu et al., 1999, Yamamoto and Sakashita, 1999, Zhao et al., 1999, Nakano et al., 2000). Immediately downstream from OFQ is a lysine–arginine processing signal, followed by a heptadecapeptide that, similar to OFQ, begins with phenylalanine and ends with glutamine; it is referred to as orphanin FQ2 (OFQ II_1–17; ppOFQ 154–170). In contrast to the nociceptive effects associated with OFQ, intracerebroventricular administration of OFQ II_1–17 is analgesic at high doses in mice (Rossi et al., 1998). Interestingly, the C-terminal extension of the OFQ II_1–17 sequence (OFQ II_1–28; ppOFQ 154–181) has been implicated in both antinociceptive and pronociceptive activities. Endogenous OFQ II_1–28 has recently been identified by HPLC in tissue homogenates from four CNS sites involved in pain modulation—amygdala, periaqueductal gray, rostroventromedial medulla, and locus coeruleus. When directly injected into the amygdala or periaqueductal gray, OFQ II_1–28 produces dose-dependent antinociception (Rossi et al., 2002). This antinociceptive effect is blocked by pretreatment with naloxone. When injected into the locus coeruleus or rostroventromedial medulla, OFQ II_1–28 is hyperalgesic and the effect is not blocked by naloxone (Rossi et al., 2002). Although OFQ II_1–17 and OFQ II_1–28 both produce antinociception, OFQ II_1–17 is less effective. It has been suggested that the C-terminal extension OFQ II_1–17 is critical for producing opposing pharmacological actions (anti- and pronociceptive) when injected into different brain sites (Rossi et al., 2002).

The CNS distributions of ORL1 and ppOFQ mRNA in the rat have been described in detail (Neal et al., 1999a, Neal et al., 1999b). Other anatomical studies of this neuropeptide system include analyses of [³H]-OFQ binding in adult mouse brain (Florin et al., 1997), [¹²⁵I]-OFQ binding in rat and human hypothalamus (Makman et al., 1997), OFQ and ORL1 mRNA distribution in developing mouse brain (Ikeda et al., 1998), and OFQ and ORL1 mRNA distribution in developing rat and human brain (Neal et al., 2001). In all anatomical studies, OFQ and ORL1 were shown to be widely distributed throughout the brain and spinal cord, with prominent localization in areas associated with pain processing, including the amygdala, periaqueductal gray, brainstem raphe nuclei, locus coeruleus, parabrachial nucleus, spinal nucleus of the trigeminal nerve, and dorsal horn of the spinal cord.

As with other G_i/o protein-coupled receptors, activation of ORL1 stimulates [³⁵S]-guanylyl-5′-O-(γ-thio)-triphosphate (GTPγS) binding (Wu et al., 1997). General descriptions of the distribution of OFQ-mediated [³⁵S]-GTPγS binding in the rat, mouse, and guinea pig brain have been reported (Sim et al., 1996, Shimohira et al., 1997, Sim and Childers, 1997). The present study was undertaken to examine the binding and functional autoradiography of ppOFQ peptide products at the opioid and ORL1 receptors. Utilizing agonist-stimulated GTPγS autoradiography (Sim et al., 1996), we have evaluated GTPγS binding in brain using OFQ, nocistatin, OFQ II_1–17, and its C-terminal extension OFQ II_1–28. At high concentrations, OFQ II_1–28 stimulated GTPγS binding in a μ-like distribution, similar to that observed with DAMGO binding. This effect is very interesting, given the analgesic properties recently demonstrated for this C-terminal peptide sequence (Rossi et al., 2002). We present additional studies with the cloned μ receptor, however, suggesting that this effect is not directly mediated by the μ opioid receptor.