Acute desensitization of nociceptin/orphanin FQ inhibition of voltage‐gated calcium channels in freshly dissociated hippocampal neurons

Abstract

Nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for opioid receptor-like receptor, has been shown to inhibit high-voltage-gated calcium channels (VGCCs) in acutely dissociated rat hippocampal pyramidal cells [Knoflach, F., Reinscheid, R.K., Civelli, O. & Kemp, J.A. (1996), J. Neurosci., 16, 6657]. In this study, it was further demonstrated that N/OFQ inhibition of calcium channel current was blocked by its specific antagonist PGN, [Phe1-psi(CH2-NH)-Gly2]nociceptin (1-13)-NH2, and the EC50 of the N/OFQ inhibition was approximately 10 nM, indicating that this effect was really mediated via the opioid receptor-like receptor. The N/OFQ inhibition of the calcium channel current was significantly reduced, as the maximal inhibition decreased from 36 to 23%, by 1-min pretreatment of freshly dissociated hippocampal neurons with the same peptide. The inhibition completely recovered from this acute desensitization in less than 20 min. The N/OFQ inhibition was also greatly attenuated by pretreatment of the neurons with the GABAB (gamma-aminobutyric acid) agonist baclofen while the baclofen inhibition of the calcium channel current was significantly reduced by N/OFQ pretreatment, revealing the agonist-induced desensitization was heterologous in nature. This desensitization was blocked by pretreating the neurons with the sodium channel blocker, tetrodotoxin (TTX), or by removing the extracellular calcium, which indicates the necessity of membrane depolarization and extracellular calcium influx in the process. Furthermore, pretreatment of the neurons with the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), attenuated the N/OFQ inhibition of the calcium channel current whereas the cAMP-dependent kinase A activator, forskolin, showed no effect, suggesting the probable involvement of PKC in the N/OFQ-induced desensitization.

Orphanin FQ/Nociceptin-Mediated Desensitization of Opioid Receptor-Like 1 Receptor and μ Opioid Receptors Involves Protein Kinase C: A Molecular Mechanism for Heterologous Cross-Talk

“…OFQ/N pretreatment did not alter basal (57.8 Ϯ 10.6 pmol/mg) or forskolin-stimulated (98.5 Ϯ 16.2 pmol/mg) levels of cAMP compared with vehicle-treated controls (basal, 64.7 Ϯ 16; forskolin, 135.6 Ϯ29 pmol/mg). Since previous reports linked ORL1 activation and desensitization with PKC (Lou et al, 1997;Pei et al, 1997;Pu et al, 1999), we explored the possibility that OFQ/N-mediated heterologous desensitization of opioid receptors also involved PKC. Cells were pretreated with the PKC inhibitor, chelerythrine (1 M), 15 min before addition of OFQ/N.…”

“…The involvement of PKC in both ORL1 and opioid receptor signaling is well documented (Chen and Yu, 1994;Lou et al, 1997;Pei et al, 1997;Pu et al, 1999), and makes PKC a likely participant in any -ORL1 receptor interaction. PKC is a serine/threonine kinase that regulates a multitude of cellular functions and is a downstream target for a plethora of agonist-mediated signal transduction events (for review see Black, 2000).…”

“…Heterologously produced phosphorylation and desensitization of receptors is also blocked by PKC inhibition (Zhang et al, 1996). OFQ/N activates PKC through ORL1 receptors (Lou et al, 1997), and homologous desensitization of ORL1 is mediated by PKC Pu et al, 1999). Since PKC is involved in the regulation of both and ORL1 receptors, its role in OFQ/N-induced and ORL1 desensitization was investigated.…”

μ-Opioid-Induced Desensitization of Opioid Receptor-Like 1 and μ-Opioid Receptors: Differential Intracellular Signaling Determines Receptor Sensitivity

“…We recently reported that prolonged morphine or DAMGO treatment up-regulates GRK2 levels in human neuroblastoma cells and contributes to ORL1 desensitization . Acute heterologous desensitization of ORL1 has been demonstrated (Pu et al, 1999), but the mechanism for this acute desensitization was not determined.…”

Orphanin FQ/Nociceptin Potentiates [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin–Induced μ-Opioid Receptor Phosphorylation

“…OFQ/N and NOP receptors are widely distributed in the central nervous system and are colocalized with receptors in many cells in the descending analgesic pathway (Heinricher et al, 1994;Connor et al, 1996;Pan et al, 2000). OFQ/N binds to NOP receptors and activates protein kinase C, which plays a role in homologous NOP receptor desensitization (Lou et al, 1997;Pei et al, 1997;Pu et al, 1999;Mandyam et al, 2002). Besides homologous NOP receptor desensitization, activation of NOP receptors by OFQ/N heterologously regulates the receptor response to DAMGO (Hawes et al, 1998;Mandyam et al, 2000Mandyam et al, , 2003Thakker and Standifer, 2002).…”