Dataset Information

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

ABSTRACT:

Background and purpose

The 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/OFQ_ATTO594 ) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression.

Experimental approach

We assessed N/OFQ_ATTO594 receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQ_ATTO594 binding was measured in (i) HEK cells expressing NOP and NOP_GFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca²⁺ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN).

Key results

N/OFQ_ATTO594 bound to NOP receptor with nM affinity and high selectivity. N/OFQ_ATTO594 activated NOP receptor by reducing cAMP formation and increasing Ca²⁺ levels in CHO_hNOPGαqi5 cells. N/OFQ_ATTO594 was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP-GFP-tagged receptors, N/OFQ_ATTO594 was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand-receptor interaction. When the NOP_GFP receptor is activated by N/OFQ_ATTO594 , movement of ligand and receptor from the cell surface to the cytosol can be measured.

Conclusions and implications

In 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/OFQ_ATTO594 to study a wide range of N/OFQ-driven cellular responses.

SUBMITTER: Bird MF

PROVIDER: S-EPMC6255954 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledNociceptin/orphanin FQ peptide (NOP) receptor is a new class of opioid receptor that may play a pathophysiologic role in anxiety and drug abuse and is a potential therapeutic target in these disorders. We previously developed a high-affinity PET ligand, (11)C-NOP-1A, which yielded promising results in monkey brain. Here, we assessed the ability of (11)C-NOP-1A to quantify NOP receptors in human brain and estimated its radiation safety profile.MethodsAfter intravenous injection of (11)C-NOP-1A, 7 healthy subjects underwent brain PET for 2 h and serial sampling of radial arterial blood to measure parent radioligand concentrations. Distribution volume (V(T); a measure of receptor density) was determined by compartmental (1- and 2-tissue) and noncompartmental (Logan analysis and Ichise's bilinear analysis [MA1]) methods. A separate group of 9 healthy subjects underwent whole-body PET to estimate whole-body radiation exposure (effective dose).ResultsAfter (11)C-NOP-1A injection, the peak concentration of radioactivity in brain was high (∼5-7 standardized uptake values), occurred early (∼10 min), and then washed out quickly. The unconstrained 2-tissue-compartment model gave excellent V(T) identifiability (∼1.1% SE) and fitted the data better than a 1-tissue-compartment model. Regional V(T) values (mL·cm(-3)) ranged from 10.1 in temporal cortex to 5.6 in cerebellum. V(T) was well identified in the initial 70 min of imaging and remained stable for the remaining 50 min, suggesting that brain radioactivity was most likely parent radioligand, as supported by the fact that all plasma radiometabolites of (11)C-NOP-1A were less lipophilic than the parent radioligand. Voxel-based MA1 V(T) values correlated well with results from the 2-tissue-compartment model, showing that parametric methods can be used to compare populations. Whole-body scans showed radioactivity in brain and in peripheral organs expressing NOP receptors, such as heart, pancreas, and spleen. (11)C-NOP-1A was significantly metabolized and excreted via the hepatobiliary route. Gallbladder had the highest radiation exposure (21 μSv/MBq), and the effective dose was 4.3 μSv/MBq.Conclusion(11)C-NOP-1A is a promising radioligand that reliably quantifies NOP receptors in human brain. The effective dose in humans is low and similar to that of other (11)C-labeled radioligands, allowing multiple scans in 1 subject.