The nociceptin/orphaninFQ (N/OFQ) peptide receptor (NOP) is a nonclassical member of the opioid receptor family. Activation of this receptor with N/OFQ produces a wide range of physiological responses and for the anesthesiologist, this receptor-transmitter system is involved in pain transmission, producing analgesia spinally and hyperalgesia/antiopioid actions in the brain.1–3 NOP receptors are also expressed in peripheral tissues, including the gastrointestinal tract and vas deferens.1–3 We recently demonstrated that peripheral blood mononuclear cells (PBMCs) from healthy volunteers transcribe mRNA encoding NOP (but not classical opioid) receptor,4 adding further detail to the neuroimmune axis.5–9 In this study, we detected mRNA encoding the precursor for pre-pro-N/OFQ (ppN/OFQ) and suggest autoregulation of lymphocyte function by N/OFQ.
This study was performed using venous blood collected from 10 healthy male volunteers (from the University Division of Anesthesia, aged 23–41 yr) with the approval of our local research ethics committee and with informed consent. Up to a maximum of 27 mL of blood was collected into EDTA-monovette tubes and diluted (1:1) with phosphate buffered saline at room temperature. This mixture was carefully layered over Ficoll-Paque Plus (Amersham Biosciences, UK; 3 mL Ficoll:4 mL blood) and centrifuged at 400g for 30 min at room temperature (18°C–20°C). PBMCs were harvested from above the Ficoll, diluted with phosphate buffered saline, and sedimented for 10 min at 100g and 4°C and ribonucleic acid (RNA) extracted using a modification of Chomczynski and Sacchi's method.4,10 Extracted RNA was finally resuspended in 40 μL of RNAse free water. Analysis for integrity and quantity yielded A260/280 >1.7 in all cases, with clear ribosomal RNA bands on gel analysis.
Reverse transcription kits from Applied Biosystems (Foster City, CA) were used to convert RNA into cDNA for use in polymerase chain reaction (PCR) according to the manufacturer's instructions. ppN/OFQ was probed with the following primers. For standard PCR the NOPP1 primer pair was used (Forward-5′-CCTGCACCATGAAAGTCCTG-3′ (exon 1), Reverse-5′-CCTTCCGGCTACACATTACC-3′ (exon 3), with an amplicon size of 546), whereas the NOPP2 primer pair was used for quantitative real-time PCR (QPCR) reactions (Forward-5′-CCTGCACCAGAATGGTAATG-3′ (exon 2), Reverse-5′-GCTGAGCACATGCTGTTTG-3′ (exon 3) amplicon size 106).
An end-point standard PCR reaction was used with an initial 95°C, 3 min denaturing step followed by 40 cycles of 95°C (30 s), 59.8°C (30 s), 72°C (30 s), then 10 min at 72°C followed by 4°C. The annealing temperature of 59.8°C was primer pair-dependent. All experiments were performed using an Eppendorf Mastercycler. PCR products were run on a 3% agarose gel stained with ethidium bromide for 45 min at 100 V and imaged under ultraviolet illumination.
Further QPCR reactions were run using SYBR green fluorescence for imaging. These reactions had a thermal profile of 95°C 10 min, 40 cycles of 95°C (30 s) 57°C (1 min) 72°C (30 s). QPCR reactions were performed using a Stratagene Mx4000 machine and inbuilt software.
All PCR assays were capable of differentiating gDNA from cDNA by virtue of primers being located on different exons. In QPCR, a deflection from the baseline before the 35th cycle of amplification was considered significant. Where appropriate, data are expressed as mean ± sem, from 10 volunteers.
In standard gel-based PCR using NOPP1 primer pairs, all 10 PBMC samples give a clear amplicon between 500 and 600 base pairs (expected amplicon for these primers 546 base pairs) (Fig. 1A). Figure 1B shows a typical growth curve for 5 of 10 PBMC samples using NOPP2 primer pairs in QPCR experiments. Cycle thresholds (which is an index of quantity of RNA present in the reaction, lower values equate to more RNA) were 30.91 ± 0.18 (n = 10). As a negative control water produced no increase in SYBR green fluorescence.
Using two PCR experimental paradigms, we have shown that PBMCs prepared from 10 healthy volunteers transcribe the precursor for N/OFQ and that these immunocytes may produce and release N/OFQ and/or other N/OFQ related peptides. The human isoform of ppN/OFQ is 176 amino acids in length and encodes at least three active peptides, N/OFQ, N/OFQ-2, and nocistatin.1 N/OFQ activates NOP to modulate a range of biological functions including nociperception. The role of N/OFQ-2 is largely unknown and nocistatin reverses the effects of N/OFQ, but not via an interaction with NOP.1,11,12 Ideally, we would have liked to probed specifically for N/OFQ, but since this arises from a precursor that is alternatively cleaved posttranscription, we could only assess ppN/OFQ. In a previous study, we examined the expression of classical (MOP-μ, DOP-Δ, and KOP-κ) and nonclassical (NOP) opioid receptors in PBMCs.4 We failed to detect classical opioid receptors; but in a similar series of PCR experiments, we did detect NOP transcripts, probably at low expression. At this point, it seems reasonable to postulate that as the PBMC express NOP receptors and the ability to produce N/OFQ, then some form of autoregulation of function may occur. In inflammation, classical opioid receptors are upregulated on peripheral nerves, and white blood cells attracted to these sites release their opioid peptides to provide a degree of endogenous analgesia via the neuroimmune axis.6 On the basis of our data, and that of others, we suggest that the NOP-N/OFQ system is part of this axis. Indeed, Fiset et al. demonstrated that human neutrophils released N/OFQ in response to fMLP and cytochalasin B.8 A note of caution should be added to these observations, since more that one peptide may be produced. If N/OFQ is released, NOP would be activated, but if nocistatin is released it is possible that N/OFQ signaling might be inhibited. The relative abundance of these peptides in stimulated samples will require measurement.
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