Effects of chronic nociceptin/orphanin FQ exposure on cAMP accumulation and receptor density in Chinese hamster ovary cells expressing human nociceptin/orphanin FQ receptors

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Abstract

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the Gi-coupled N/OFQ receptor (NOP). We have examined the effects of chronic exposure of Chinese hamster ovary cells expressing the recombinant human NOP receptor (CHOhNOP) to 1 nM N/OFQ for up to 48 h in the absence and presence of the NOP selective antagonist [Nphe1]N/OFQ (1–13)NH2 ([Nphe1]). Then, either a concentration–response curve for N/OFQ inhibition of cAMP formation was constructed or the cells were homogenized and membrane receptor density was determined using [125I]Y14N/OFQ. There was a time-dependent reduction in pEC50 (without a change in maximum) for N/OFQ with significant differences observed following >24 h of exposure (control pEC50∼9.5; 48 h pretreatment∼8.7). In cells co-exposed to N/OFQ+[Nphe1] for 48 h, there was no reduction in pEC50. There was a compensatory (∼2.5-fold), [Nphe1]-sensitive increase in cAMP mass in cells exposed to N/OFQ for 24–48 h. N/OFQ pretreatment also resulted in a time-dependent [Nphe1]-sensitive loss of cell surface receptors. At 48 h, Bmax was reduced from ∼2.0 to ∼1.3 pmol mg−1 protein without a change in pKd for N/OFQ. There was a positive correlation between pEC50 for cAMP inhibition and Bmax. The lack of effect on maximum cAMP response probably results from receptor overexpression and the creation of a receptor reserve.

Introduction

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the opioid receptor like receptor-1 Meunier et al., 1995, Reinscheid et al., 1995, hereafter referred to as the N/OFQ receptor (NOP) (Cox et al., 2000). Based on structural and coupling similarities, the N/OFQ–NOP receptor system is considered to be a novel member of the opioid receptor family Calo' et al., 2000b, Mogil and Pasternak, 2001. Activation of this Gi-coupled “inhibitory” receptor produces an inhibition in the formation of cAMP, closure of voltage-sensitive Ca2+ channels, and stimulation of an outward K+ conductance in a variety of preparations Hawes et al., 2000, Calo' et al., 2000b, Meunier, 2000, Mogil and Pasternak, 2001. At a systems level, activation of this receptor produces a diverse array of physiological responses including modulation of nociception, feeding, cardiovascular system, and the production of anxiolysis Calo' et al., 2000b, Meunier, 2000, Mogil and Pasternak, 2001. If this system is to be utilized with effect in man, then it is necessary to characterize and understand the response to continued activation, i.e., possible desensitization.

Continuous exposure to agonist results in loss of agonist responsiveness (Lohse, 1993). Indeed, exposure of classical opioid receptors to opioid agonists results in reduced agonist responsiveness with receptor–G protein uncoupling or loss of membrane-bound receptors resulting in a diminished ability to inhibit cAMP accumulation. This has been demonstrated for μ-opioid (MOP) (e.g., Yabaluri and Medzihradsky, 1997), δ-opioid (DOP) (e.g., Allouche et al., 1999), and κ-opioid (KOP) receptors (e.g., Raynor et al., 1994). Consistent with these observations for classical opioid receptors, several studies have been performed with NOP (Hawes et al., 2000). Acute exposure of NOP to N/OFQ results in attenuation of N/OFQ-induced: (a) increase in extracellular acidification in Chinese hamster ovary (CHO) cells stably expressing human NOP (Pei et al., 1997), (b) inhibition of cAMP formation in human neuroblastoma SK-N-SH cells (Cheng et al., 1997), and in NG108-15 hybrid cells (Ma et al., 1997), (c) inhibition of Ca2+ channel currents in NG108-15 cells (Morikawa et al., 1998), (d) increase in inwardly-rectifying K+ currents in locus caeruleus neurons (Connor et al., 1996), and (e) activation of mitogen-activated protein kinase (Hawes et al., 1998). The effects of chronic N/OFQ exposure are largely unknown.

In the present study, we examined the effects of chronic (up to 48 h) N/OFQ exposure in CHO cells expressing the recombinant human NOP receptor (CHOhNOP). As a measure of receptor activity/desensitization, we monitored the well-described inhibition of forskolin-stimulated cAMP formation and as a crude measure of down-regulation loss of [125I]Y14N/OFQ binding sites. In addition, we have utilized the highly selective and competitive NOP antagonist [Nphe1]N/OFQ (1–13)NH2 ([Nphe1]) in these studies.

Section snippets

Sources of chemicals and reagents

N/OFQ and [Nphe1] were synthesized at one of our institutes. All cell culture media and supplements were from Life Technologies (Paisley, UK). [2,8-3H]-cAMP (28.4 Ci·mmol−1) and [125I]Y14N/OFQ (2000 Ci·mmol−1) were from NEN DuPont (Boston, MA) and Amersham (Little Chalfont, UK), respectively. Phosphoramidon was from peptide institute (Osaka, Japan). Amastatin, bestatin, and captopril were from Sigma (Poole, UK). All other reagents were of the highest purity available. CHO cells stably

Preliminary studies

As it is possible that N/OFQ may “stick” in our desensitizing challenges, we initially determined how much of a 1 nM N/OFQ solution, spiked with 15 pM of [125I]Y14N/OFQ and incubated for 2 h, could be washed away using the protocol described in Methods. [125I]Y14N/OFQ present on or in the harvested cells was assessed using a Gamma counter. The residual activity of incubated N/OFQ was up to 3.7±0.3% of control (results from four different experiments) equivalent to ∼30 pM. At this concentration,

Discussion

In this study utilizing CHOhNOP cells, we have shown that acute (up to 6 h) treatment with 1 nM N/OFQ is not sufficient to induce significant desensitization of response at the level of adenylyl cyclase or loss of cell surface receptors. However, chronic (>24 h) treatment results in a rightward shift in the concentration–response curve to subsequent N/OFQ rechallenge, an up-regulation of cAMP formation, and loss of cell surface receptors. The rightward shift in the cAMP concentration–response

Acknowledgements

We would like to thank Dr. F. Marshall and Mrs. N. Bevan of Glaxo-Wellcome for providing CHO cells expressing the human N/OFQ receptor.

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