Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594: a novel fluorescent probe for the N/OFQ (NOP) receptor

Abstract

Background and PurposeThe nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore‐ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression.Experimental ApproachWe assessed N/OFQATTO594 receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQATTO594 binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN).Key ResultsN/OFQATTO594 bound to NOP receptor with nM affinity and high selectivity. N/OFQATTO594 activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPGαqi5 cells. N/OFQATTO594 was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP‐GFP‐tagged receptors, N/OFQATTO594 was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand–receptor interaction. When the NOPGFP receptor is activated by N/OFQATTO594, movement of ligand and receptor from the cell surface to the cytosol can be measured.Conclusions and ImplicationsIn the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQATTO594 to study a wide range of N/OFQ‐driven cellular responses.

Opioid Receptors in Immune and Glial Cells—Implications for Pain Control

“…The sequencing of PCR products revealed that NOP receptor transcripts from immune cells and brain were identical (115,116). Some studies detected the NOP receptor mRNA transcripts in leukocytes at levels comparable to those in human cerebral cortex (115), whereas others found them at very low amounts (88,121). Together, opioid receptor mRNAs appear to be expressed at relatively low levels in immune cells as compared to neuronal tissue or opioid receptor-expressing immune cell lines (88-91, 109, 110, 121, 122).…”

“…NOP receptor protein detection appears variable, with high affinity radiolabeled N/OFQ binding in granulocytes (116), but lack of such binding in PBMC (88) from human blood. The latter research group recently detected NOP receptor binding (reversible by selective NOP receptor antagonist SB-612111) in human blood granulocytes using a novel fluorescent probe for the receptor (a red fluorophore-ATTO594 conjugated to the N/OFQ) (121).…”

Functional Selectivity Does Not Predict Antinociceptive/Locomotor Impairing Potencies of NOP Receptor Agonists

“…Collectively these results can be interpreted assuming that βarr2 acts [as demonstrated for several GPCRs ( Gurevich and Gurevich, 2019 )] as a desensitizing element of the antinociceptive response to mu opioid as well as NOP receptor agonists. Of note, NOP receptor phosphorylation and internalization have already been described by different research groups ( Spampinato et al, 2001 ; Corbani et al, 2004 ; Zhang et al, 2012 ; Bird et al, 2018 ; Mann et al, 2019 ). βarr2( − / − ) mice were slightly less sensitive than βarr2(+/+) mice to the effects of Ro 65-6570 in the rotarod, with the 3 mg/kg dose being active in wild-type animals but not in mice lacking the βarr2 gene.…”

“…Male CD-1 mice (ENVIGO, Udine, Italy) were used in this study together with mice knockout for the NOP receptor gene [NOP(−/−)] and βarr2(+/+) and βarr2(−/−) mice. Details about the generation of NOP(−/−) mice have been published previously (Nishi et al, 1997;Bertorelli et al, 2002), moreover NOP(+/+) and NOP(−/−) mice have been backcrossed on CD-1 strain in our laboratories. βarr2(−/−) mice were from The Jackson Laboratory [JAX stock #011130; (Bohn et al, 1999)] and C57BL/6J mice were used as controls.…”

Potential Role of SLC5A8 Expression in the Etiology of Subacute Ruminal Acidosis

“…Then the ruminal epithelium cells were incubated with goat anti-rabbit IgG conjugated with FITC (1:200; Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 30 min and counterstained with Hoechst 33258 (Beyotime) ( 23 ). Finally, the coverslips (YA0774; Solarbio) were sealed with glycerol (MB9893; Meilun), and the samples were observed by laser confocal microscopy (FV500, OLYMPUS, China) ( 24 ).…”