Nociceptin receptor-mediated Ca2+ channel inhibition and its desensitization in NG108-15 cells

doi:10.1016/S0014-2999(98)00306-9

European Journal of Pharmacology

Volume 351, Issue 2, 19 June 1998, Pages 247-252

https://doi.org/10.1016/S0014-2999(98)00306-9 Get rights and content

Abstract

It has been shown that the membrane of hybrid NG108-15 neuroblastoma×glioma cells contains a high-affinity binding site for nociceptin. In the present study, we first demonstrated the expression of nociceptin receptor mRNA in NG108-15 cells. Application of nociceptin to NG108-15 cells produced a concentration-dependent (EC₅₀=29 nM) inhibition of Ca²⁺ channel currents in a pertussis toxin-sensitive fashion. This nociceptin-induced inhibition of Ca²⁺ channel currents was prevented in the presence of ω-conotoxin GVIA, a blocker of the N-type Ca²⁺ channel, and had both voltage-dependent and -independent components. Prolonged application of nociceptin elicited homologous desensitization of the inhibition with a time constant of 5.3 min. These results indicate that the nociceptin receptor is coupled to the N-type Ca²⁺ channel via pertussis toxin-sensitive G proteins in NG108-15 cells and that this coupling is associated with rapid and homologous desensitization.

Introduction

During the course of attempts to clone the cDNAs of three major types of opioid receptors (μ, δ and κ), our laboratory and others have identified a novel member of the opioid receptor family, termed ROR-C (Fukuda et al., 1994), ORL1 (Mollereau et al., 1994) or LC132 (Bunzow et al., 1994). Although it has >50% amino acid sequence homology with μ, δ and κ opioid receptors, none of the opioid receptor ligands tested bind to it with high affinity. A heptadecapeptide has been isolated from mammalian brain as an endogenous ligand for this `orphan' receptor and was named nociceptin (Meunier et al., 1995) or orphanin FQ (Reinscheid et al., 1995). In contrast to the analgesic effect produced by opioid receptor agonists, nociceptin is reported to induce hyperalgesia when injected intracerebroventricularly (Meunier et al., 1995; Reinscheid et al., 1995). At the cellular level, however, application of nociceptin elicits similar effects as those induced by activation of opioid receptors, such as inhibition of adenylate cyclase (Meunier et al., 1995; Reinscheid et al., 1995) and voltage-gated Ca²⁺ channels (Connor et al., 1996a; Knoflach et al., 1996) or activation of inwardly rectifying K⁺ channels (Vaughan and Christie, 1996; Connor et al., 1996b; Vaughan et al., 1997) and mitogen-activated protein kinase (MAPK; Fukuda et al., 1997).

NG108-15 mouse neuroblastoma×rat glioma hybrid cells possess a homogeneous population of δ-opioid receptors (Evans et al., 1992) and have been used extensively to study the cellular effects of opioids. The δ-opioid receptor is negatively coupled to adenylate cyclase and the N-type Ca²⁺ channel via pertussis toxin-sensitive G proteins in NG108-15 cells (Morikawa et al., 1995). It has been shown recently that nociceptin binds to the NG108-15 cell membrane with high affinity and inhibits adenylate cyclase in NG108-15 cells (Ma et al., 1997). In the present investigation, we demonstrate the expression of nociceptin receptor mRNA, using the rat nociceptin receptor (ROR-C) cDNA probe, and the coupling of the nociceptin receptor to the N-type Ca²⁺ channel in NG108-15 cells. We further show that this nociceptin receptor-mediated Ca²⁺ channel inhibition undergoes rapid and homologous desensitization.

Section snippets

Drugs

Nociceptin was obtained from TOCRIS COOKSON (St. Louis, MO, USA). [d-Ala², d-Leu⁵]enkephalin (DADLE) was purchased from Peptide Institute (Osaka, Japan). [ $α-^{32} P$ ]dCTP was from Amersham (Tokyo, Japan). All other chemicals were obtained from Wako Pure Chemical Industries (Osaka, Japan), Nacalai Tesque (Kyoto, Japan) and Sigma (St. Louis, MO, USA).

Cell culture

NG108-15 cells were cultured as described previously (Morikawa et al., 1998). For electrophysiological recordings, cells were plated onto 15-mm-diameter

Expression of nociceptin receptor mRNA in NG108-15 cells

In order to examine the expression of nociceptin receptor mRNA in NG108-15 cells, poly(A)⁺ RNA preparations from NG108-15 cells were subjected to blot hybridization analysis, using the ROR-C cDNA probe (Fig. 1). NG108-15 cells contained an RNA species of ∼3.7 kb that hybridized with the ROR-C cDNA probe and corresponded in size to the major RNA species detected in rat cerebrum.

Nociceptin receptor-mediated inhibition of Ca²⁺ channel currents in NG108-15 cells

We next tested whether the nociceptin receptor expressed in NG108-15 cells was functionally coupled to Ca²⁺ channels.

Discussion

We detected the expression of nociceptin receptor mRNA in NG108-15 cells by RNA blot hybridization analysis, using the rat nociceptin receptor (ROR-C) cDNA probe. This strongly suggests that ROR-C or its mouse counterpart represents the high-affinity binding site for nociceptin in NG108-15 cells reported recently (Ma et al., 1997).

Nociceptin rapidly and potently inhibited Ca²⁺ channel currents in NG108-15 cells, with an EC₅₀ of 29 nM. The potency of nociceptin observed here is comparable to the

Acknowledgements

We thank Natsumi Kikkawa for technical assistance. This work was supported by research grants from the Ministry of Education, Science and Culture of Japan.

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Cited by (28)

Helix-Constrained Nociceptin Peptides Are Potent Agonists and Antagonists of ORL-1 and Nociception
2015, Vitamins and Hormones
Citation Excerpt :
The most attractive method to test compounds is therefore to develop an assay using a nontransfected native, yet immortalized, cell line. ORL-1 has been identified to be present on a number of immune and neuronal immortalized cell lines, including U937 and THP-1 (monocytes; Peluso et al., 2001, 1998), EBV 1–4 (B cells; Peluso et al., 2001, 1998), MOLT-4 (T cells; Peluso et al., 2001, 1998), SH-SY5Y (neuroblastoma; Cheng, Standifer, Tublin, Su, & Pasternak, 1995; Peluso et al., 2001), SK-N-SH (neuroblastoma; Cheng et al., 1997), SK-N-BE (neuroblastoma; Spampinato, Di Toro, & Qasem, 2001), and NG108-15 (neuroblastoma × glioma; Morikawa et al., 1998). The ORL-1 receptor has also been found on primary immune cell lines (peripheral blood lymphocytes (Arjomand, Cole, & Evans, 2002; Pampusch et al., 2000; Peluso et al., 1998), PMNs (Fiset, Gilbert, Poubelle, & Pouliot, 2003; Peluso et al., 1998), and macrophages (Arjomand et al., 2002; Pampusch et al., 2000)) as well as on nervous system cell types (rat neurons (Buzas, Rosenberger, & Cox, 1998) and astrocytes (Buzas, Rosenberger, Kim, & Cox, 2002; Buzas, Symes, & Cox, 1999; Takayama & Ueda, 2005)).
Nociceptin (orphanin FQ) is a 17-residue neuropeptide hormone with roles in both nociception and analgesia. It is an opioid-like peptide that binds to and activates the G-protein-coupled receptor opioid receptor-like-1 (ORL-1, NOP, orphanin FQ receptor, kappa-type 3 opioid receptor) on central and peripheral nervous tissue, without activating classic delta-, kappa-, or mu-opioid receptors or being inhibited by the classic opioid antagonist naloxone. The three-dimensional structure of ORL-1 was recently published, and the activation mechanism is believed to involve capture by ORL-1 of the high-affinity binding, prohelical C-terminus. This likely anchors the receptor-activating N-terminus of nociception nearby for insertion in the membrane-spanning helices of ORL-1. In search of higher agonist potency, two lysine and two aspartate residues were strategically incorporated into the receptor-binding C-terminus of the nociceptin sequence and two Lys(i) → Asp(i + 4) side chain–side chain condensations were used to generate lactam cross-links that constrained nociceptin into a highly stable α-helix in water. A cell-based assay was developed using natively expressed ORL-1 receptors on mouse neuroblastoma cells to measure phosphorylated ERK as a reporter of agonist-induced receptor activation and intracellular signaling. Agonist activity was increased up to 20-fold over native nociceptin using a combination of this helix-inducing strategy and other amino acid modifications. An NMR-derived three-dimensional solution structure is described for a potent ORL-1 agonist derived from nociceptin, along with structure–activity relationships leading to the most potent known α-helical ORL-1 agonist (EC₅₀ 40 pM, pERK, Neuro-2a cells) and antagonist (IC₅₀ 7 nM, pERK, Neuro-2a cells). These α-helix-constrained mimetics of nociceptin(1–17) had enhanced serum stability relative to unconstrained peptide analogues and nociceptin itself, were not cytotoxic, and displayed potent thermal analgesic and antianalgesic properties in rats (ED₅₀ 70 pmol, IC₅₀ 10 nmol, s.c.), suggesting promising uses in vivo for the treatment of pain and other ORL-1-mediated responses.
Two C-terminal amino acids, Ser334 and Ser335, are required for homologous desensitization and agonist-induced phosphorylation of opioid receptor-like 1 receptors
2006, Cellular Signalling
Various cellular signaling pathways induced by nociceptin activation of ORL1 (opioid receptor-like 1 receptor) develop homologous desensitization. Multiple lines of evidence suggest that agonist-induced phosphorylation of serine (Ser)/threonine (Thr) residues at intracellular carboxyl tail leads to homologous desensitization of G protein-coupled receptors. In the present study, we investigated the functional role played by C-terminal Ser/Thr residues in agonist-induced desensitization and phosphorylation of ORL1. In HEK 293 cells expressing wild-type ORL1 and ORL1(CΔ21), which lacks C-terminal 21 amino acids, nociceptin inhibition of adenylate cyclase activity exhibited homologous desensitization after 1 h pretreatment of nociceptin. In contrast, ORL1(CΔ34), which differs with ORL1(CΔ21) by lacking C-terminal Ser³³⁴, Ser³³⁵ and Ser³⁴³ residues, failed to develop agonist-induced desensitization. Point mutant (S343A) ORL1 underwent homologous desensitization after nociceptin pretreatment. Substituting Ser³³⁴ or Ser³³⁵ with alanine greatly impaired nociceptin-induced ORL1 desensitization. In HEK 293 cells expressing double mutant (S334A/S335A) ORL1, nociceptin pretreatment failed to significantly affect the efficacy and potency by which nociceptin inhibits forskolin-stimulated cAMP formation. Mutation of Ser³³⁴ and Ser³³⁵ also greatly reduced nociceptin-induced ORL1 phosphorylation. These results suggest that two C-terminal serine residues, Ser³³⁴ and Ser³³⁵, are required for homologous desensitization and agonist-induced phosphorylation of ORL1.
Effects of intraplantar injections of nociceptin and its N-terminal fragments on nociceptive and desensitized responses induced by capsaicin in mice
2005, Peptides
Injection of capsaicin into the hindpaw has been employed as a model of chemogenic nociception in mice. Intraplantar injection of nociceptin (30–240 pmol) produced a significant and dose-dependent antinociceptive activity in the capsaicin test. The nociceptin N-terminal fragments, (1–11) and (1–13), were also active with a potency higher than nociceptin and comparable to nociceptin, respectively. Intraplantar injection of the nociceptin (1–7) fragment had no effect on capsaicin-induced nociception. Antinociception induced by nociceptin or nociceptin (1–13) was reversed significantly by intraplantar co-injection of [Nphe¹]nociceptin (1–13)NH₂, an orphan opioid receptor-like 1 (ORL1) receptor antagonist, whereas local injection of the antagonist did not interfere with the action of nociceptin (1–11). Nociceptin (1–11) was approximately 2.0-fold more potent than naturally occurring peptide nociceptin, and 10-fold more active than intraplantar morphine. Nociceptive licking/biting response to intraplantar injection of capsaicin was desensitized by repeated injections of capsaicin at the interval of 15 min. Desensitization induced by capsaicin was attenuated significantly by co-injection of nociceptin at much lower doses than antinociceptive ED₅₀ for nociceptin. Capsaicin desensitization was also decreased by co-injection of nociceptin (1–11) and (1–13) to a similar extent. The present results indicate that not only nociceptin but also the N-terminal fragment (1–13) possesses a local peripheral antinociceptive action, which may be mediated by peripheral ORL1 receptors. In addition, the difference of the effective doses suggests that the antinociceptive action and inhibition of capsaicin-induced desenitization by nociceptin, nociceptin (1–11) and (1–13), may involve distinct mechanisms at the level of the peripheral nerve terminal.
Effects of chronic nociceptin/orphanin FQ exposure on cAMP accumulation and receptor density in Chinese hamster ovary cells expressing human nociceptin/orphanin FQ receptors
2002, European Journal of Pharmacology
Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the G_i-coupled N/OFQ receptor (NOP). We have examined the effects of chronic exposure of Chinese hamster ovary cells expressing the recombinant human NOP receptor (CHO_hNOP) 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, B_max was reduced from ∼2.0 to ∼1.3 pmol mg⁻¹ protein without a change in pK_d for N/OFQ. There was a positive correlation between pEC₅₀ for cAMP inhibition and B_max. The lack of effect on maximum cAMP response probably results from receptor overexpression and the creation of a receptor reserve.
Nociceptin is a potent inhibitor of N-type Ca2+ channels in rat sympathetic ganglion neurons
2000, Neuroscience Letters
Nociceptin, an endogenous agonist of the opioid receptor-like₁ (ORL₁) receptor, is implicated in a wide range of physiological functions including cardiovascular control. However, the effect of nociceptin on peripheral sympathetic ganglion neurons has not been studied. Whole-cell voltage clamp was used to study Ca²⁺ currents on freshly dissociated sympathetic superior cervical ganglion neurons from juvenile rats. Nociceptin (1 μM) caused a fast inhibition of the peak currents by 69±3% in all neurons. Strong positive prepulses counteracted the inhibition of the peak current by 64% and no effect of nociceptin was observed when the cells were pre-incubated with Pertussis toxin. The inhibition was reversible and dose-dependent with an EC₅₀ of 508±50 pM. Blockade of N-type channels by 1 μM ω-conotoxin GVIA reduced the peak currents by 83±1% and abolished the action of nociceptin. Naloxone could not prevent the inhibition by nociceptin and [D-Ala², N-Me-Phe⁴, Gly⁵-ol] enkephalin (DAMGO) only depressed a small proportion of the current in 1/7 neurons. These data suggests that nociceptin inhibits transmitter release from sympathetic neurons by a selective blockade of N-type channels, which may be of importance for its depressive effect on the cardiovascular system.
Cellular neurophysiological actions of nociceptin/orphanin FQ
2000, Peptides
Cellular actions of nociceptin/orphanin FQ (N/OFQ) resemble those of μ-, δ-, and κ-opioids, i.e. activation of inwardly rectifying K⁺ conductance, inhibition of high-voltage-activated Ca²⁺ channel currents, and impediment of neurotransmitter release. Differences in ORL₁ and μ-receptor distribution lead to: 1) more widespread actions of N/OFQ on periaqueductal gray neurons than opioids and 2) differential effects of N/OFQ and opioids in the brainstem. Also, unlike opioids, N/OFQ inhibits T-type Ca²⁺ channel current in sensory neurons. Opioids and N/OFQ may modulate glutamate responses in different ways, and certain actions of N/OFQ are potentiated following nerve injury whereas those of μ-opioids are attenuated. Agonists at ORL₁ receptors may therefore be of clinical interest in the management of neuropathic pain.

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Nociceptin receptor-mediated Ca2+ channel inhibition and its desensitization in NG108-15 cells

Abstract

Introduction

Section snippets

Drugs

Cell culture

Expression of nociceptin receptor mRNA in NG108-15 cells

Nociceptin receptor-mediated inhibition of Ca2+ channel currents in NG108-15 cells

Discussion

Acknowledgements

FEBS Lett.

Neuron

Trends Neurosci.

FEBS Lett.

FEBS Lett.

FEBS Lett.

FEBS Lett.

Neuroscience

The effect of nociceptin on Ca2+ channel current and intracellular Ca2+ in the SH-SY5Y human neuroblastoma cell line

Br. J. Pharmacol.

Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro

Br. J. Pharmacol.

Cloning of a delta opioid receptor by functional expression

Science

Depression of glutaminergic transmission by nociceptin in the neonatal rat hemisected spinal cord preparation in vitro

Br. J. Pharmacol.

Inhibition of tachykinin release from peripheral endings of sensory nerves by nociceptin, a novel opioid peptide

Br. J. Pharmacol.

Inhibition by nociceptin of neurogenic inflammation and the release of SP and CGRP from sensory nerve terminals

Br. J. Pharmacol.

Nociceptin receptor-mediated Ca²⁺ channel inhibition and its desensitization in NG108-15 cells

Nociceptin receptor-mediated inhibition of Ca²⁺ channel currents in NG108-15 cells

The effect of nociceptin on Ca²⁺ channel current and intracellular Ca²⁺ in the SH-SY5Y human neuroblastoma cell line