Elsevier

Molecular Brain Research

Volume 105, Issues 1–2, 30 September 2002, Pages 38-46
Molecular Brain Research

Research report
Orphanin FQ/nociceptin blocks chronic morphine-induced tyrosine hydroxylase upregulation

https://doi.org/10.1016/S0169-328X(02)00390-XGet rights and content

Abstract

The recently discovered endogenous peptide orphanin FQ/nociceptin (OFQ/N) activates the opioid receptor-like 1 (ORL1) receptor and produces diverse effects on pain perception. In addition to producing spinal analgesia, OFQ/N also exhibits an ‘anti-opioid activity’ against functional (supraspinal analgesia) and behavioral (conditioned place preference and withdrawal) properties of morphine. One manifestation of the behavioral changes resulting from chronic use of morphine is the upregulation of tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamine biosynthesis), which contributes to the dramatic increases in catecholamine release in the target regions of the locus coeruleus (LC) and the ventral tegmental area (VTA). The present study sought to determine the molecular mechanism(s) by which OFQ/N modulates the chronic actions of morphine by utilizing human neuroblastoma cell lines [BE(2)-C and SH-SY5Y] that endogenously express TH, and μ and ORL1 receptors. Activation of μ or ORL1 receptors in these cells in turn activates extracellular signal-regulated protein kinases (ERKs), ERK1 and ERK2. Chronic activation of μ, but not ORL1, receptors upregulated TH levels in these cells as previously reported in rat brain. Morphine-induced TH upregulation was blocked upon inclusion of a MEK-1 (mitogen-activated protein kinase kinase-1) inhibitor (PD98059), confirming the role for ERKs in this adaptive response to morphine. Inclusion of OFQ/N during chronic morphine exposure also blocked morphine-induced TH upregulation. Furthermore, chronic OFQ/N exposure increased levels of the TH gene repressor, Oct-2, irrespective of the presence or absence of morphine. This report suggests a potential role for Oct-2 in mediating the anti-opioid actions of OFQ/N against the behavioral manifestations resulting from chronic use of morphine.

Introduction

Orphanin FQ/Nociceptin (OFQ/N) displays high affinity for the opioid receptor-like 1 (ORL1) receptor, but very low affinity for μ, δ or κ receptors [39]. ORL1 receptor activation by OFQ/N initiates pertussis toxin-sensitive intracellular events including inhibition of adenylyl cyclase, activation of extracellular signal-regulated kinases (ERKs), ERK1 and ERK2, and modulation of calcium and potassium channel conductances [20]. OFQ/N potently produces analgesia after spinal [20] as well as supraspinal administration [41], however it exhibits ‘anti-opioid activity’ supraspinally by antagonizing μ-, δ- and κ-opioid receptor-mediated analgesia [30].

The ‘anti-μ opioid’ actions of OFQ/N are not limited to blockade of morphine analgesia, as supraspinal OFQ/N also inhibits morphine-induced conditioned place preference [6], [32], suggesting that OFQ/N blocks the positive reinforcing effects of morphine. The establishment of conditioned place preference involves facilitation of dopaminergic transmission in the nucleus accumbens [44], [14]. Chronic morphine administration increases mesolimbic dopaminergic transmission by upregulating tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamine biosynthesis) in the neurons of the ventral tegmental area (VTA) [2], [5]. Chronic morphine-induced TH upregulation in the locus coeruleus (LC) is implicated in morphine dependence and withdrawal, where this upregulation could increase the capacity of LC neurons to synthesize and release norepinephrine [5], [16], [26]. Interestingly, OFQ/N also has been found to inhibit morphine withdrawal [23], [25].

As ORL1 and μ opioid receptors are both densely expressed [33] and co-expressed [10] on LC neurons, and ORL1 mRNA is found in TH-containing neurons of the VTA and substantia nigra [34], we hypothesized that the ability of OFQ/N to block the reinforcing and withdrawal effects of morphine may result from its ability to block morphine-induced TH upregulation. Two different human neuroblastoma cell lines, BE(2)-C and SH-SY5Y, endogenously expressing TH [8], and ORL1 [28], [9], [38] and μ opioid receptors [45], [22] were utilized to determine the mechanism(s) by which OFQ/N could modulate the intracellular changes resulting from chronic morphine.

One of the mechanisms by which chronic administration of morphine in rats produces TH upregulation, particularly in the VTA, is via activation of ERKs [3]. ERK levels are elevated in the LC and caudate putamen following chronic morphine exposure in rats [36]. Furthermore, naloxone-precipitated withdrawal in morphine-dependent rats stimulates ERK activation in the LC and the nucleus of the solitary tract, brain regions associated with the aversive components of the opioid withdrawal syndrome [42]. These studies strongly suggest an important role for ERKs in mediating the long-term adaptations in the brain that result in profound behavioral effects following chronic morphine exposure. Similar to those in vivo studies, we find that ERKs also mediate chronic morphine-induced TH upregulation in human neuroblastoma cells wherein natively expressed μ and ORL1 receptors are functionally coupled to the activation of ERKs. Besides directly interfering with the pathway through which morphine stimulates TH upregulation, the other possible mechanism for a negative modulation of TH is via induction of Oct-2 protein. Oct-2 is a POU (Pit-Oct-Unc) family transcription factor that binds to the heptamer sequence 5′-CTAATGG-3′ in the TH promoter to repress TH gene expression and reduce the endogenous levels of TH in neuronal cells [11]. The present study suggests that OFQ/N blocks chronic morphine-induced TH upregulation by upregulating the levels of Oct-2 protein.

Section snippets

Materials

The following drugs and materials were purchased from, or kindly provided by, the sources indicated: cell culture media, penicillin G, streptomycin sulfate (Gibco BRL, Grand Island, NY, USA); fetal bovine sera (Atlanta Biologicals, Norcross, GA, USA); OFQ/N, PL-017 and morphine sulfate (Research Technology Branch of the National Institute on Drug Abuse, Rockville, MD, USA); peptide III BTD (Neosystem, Strasbourg, France); PD98059, phospho-specific ERK1 and ERK2, and ERK1 and ERK2 antisera (Cell

μ and ORL1 receptor-mediated activation of ERK

To illustrate the functional coupling of natively expressed μ and ORL1 receptors to ERK activation in human neuroblastoma cells, phospho-specific ERK antiserum was used for immunoblotting. This antiserum recognizes ERK1 and ERK2 only when the enzymes are phosphorylated at both tyrosine and threonine residues, representing the fully activated state of ERKs [40]. Cells were serum-deprived for 24 h to decrease basal ERK activation in order to facilitate the quantification of opioid-induced ERK

Discussion

Recent studies have unveiled the differential role of OFQ/N in modulating the long-term deleterious effects of morphine [20], clearly demonstrating the inhibitory effect of OFQ/N on morphine-induced conditioned place preference and withdrawal [6], [32], [23]. Although ORL1 and μ opioid receptors are co-localized on several populations of neuronal cells, including the locus coeruleus [10], very little is known about how morphine and OFQ/N may modulate each others actions at the cellular level [7]

Acknowledgements

This work was supported in part, by grants from the US Public Health Service (DA10738) and the Texas Advanced Research Program (003652-0114-1999) to K.M.S.

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