The Identification of Opioid Receptors and Peptide Precursors in Human DRG Neurons Expressing Pain-Signaling Molecules Confirms Their Potential as Analgesic Targets

Abstract

The presence and function of the opioidergic system in sensory dorsal root ganglia (DRG) was demonstrated in various animal models of pain. To endorse recent functional and transcriptional evidence of opioid receptors in human DRG, this study compared morphological and transcriptional evidence in human and rat DRG using immunofluorescence confocal microscopy and mRNA transcript analysis. Specifically, it examined the neuronal expression of mu (MOR), delta (DOR), and kappa (KOR) opioid receptors, opioid peptide precursors (POMC, PENK, and PDYN), and key pain-signaling molecules. The results demonstrate abundant immunoreactivity in human DRG for key pain transduction receptors, including the thermosensitive ion channels TRPV1, TRPV4 and TRPA1, mechanosensitive PIEZO1 and PIEZO2, and the nociceptive-specific Nav1.8. They colocalized with calcitonin gene-related peptide (CGRP), a marker for peptidergic sensory neurons. Within this same subpopulation, we identified MOR, DOR, and KOR, while their ligand precursors were less abundant. Notably, the mRNA transcripts of MOR and PENK in human DRG were highest among the opioid-related genes; however, they were considerably lower than those of key pain-signaling molecules. These findings were corroborated by functional evidence in demonstrating the fentanyl-induced inhibition of voltage-gated calcium currents in rat DRG, which was antagonized by naloxone. The immunohistochemical and transcriptional demonstration of opioid receptors and their endogenous ligands in both human and rat DRG support recent electrophysiologic and in situ hybridization evidence in human DRG and confirms their potential as analgesic targets. This peripherally targeted approach has the advantage of mitigating central opioid-related side effects, endorsing the potential of future translational pain research from rodent models to humans.

Keywords: human; opioid peptides; opioid receptors; pain; peripheral sensory neurons.

Figure 1

Detection of the heat-sensitive TRPV1, TRPV4, and cold-sensitive TRPA1 pain transduction receptors in human versus rat sensory DRG neurons. TRPV1, TRPV4, and TRPA1 were visualized by specific antibodies (see Supplementary Table S3) and respective secondary antibodies labelled with Texas red fluorescence, while the sensory neuron marker CGRP was visualized by FITC green fluorescence. Both in human (left panel) as well as in rat (right panel) DRG neurons, TRPV1, TRPV4, and TRPA1 immunoreactivity is clearly detectable and colocalizes (yellow fluorescence) abundantly with the sensory neuron marker CGRP. Bars represent 55 µm.

Figure 2

Detection of the mechanosensitive ion channels PIEZO1 and PIEZO2, as well as the TTX-resistant voltage-gated Nav1.8 ion channel, in human versus rat sensory DRG neurons. PIEZO1, PIEZO2, and Nav1.8 were visualized by specific antibodies (see Supplementary Table S3) and respective secondary antibodies labelled with Texas red fluorescence, while the sensory neuron marker CGRP was visualized by FITC green fluorescence. Both in human (left panel) as well as in rat (right panel) DRG neurons, PIEZO1, PIEZO2, and Nav1.8 immunoreactivity is clearly detectable and colocalizes (yellow fluorescence) in part with the sensory neuron marker CGRP. Bars represent 55 µm.

Figure 3

Colocalization of the opioid receptor MOR together with the peripheral sensory neuron marker CGRP or the nociceptive neuron marker Nav1.8 in human versus rat sensory DRG neurons. MOR was visualized by a specific antibody (see Supplementary Table S3) and respective secondary antibody labelled with Texas red fluorescence, while CGRP or Nav1.8 was visualized by FITC green fluorescence. Both in human (left panel) as well as in rat (right panel) DRG neurons, MOR immunoreactivity colocalized (yellow fluorescence) with CGRP or Nav1.8. Bars represent 55 µm.

Figure 4

Detection of the opioid receptors KOR and DOR in human versus rat sensory DRG neurons. KOR and DOR were visualized by specific antibodies (see Supplementary Table S3) and respective secondary antibodies labelled with Texas red fluorescence, while the sensory neuron marker CGRP was visualized by FITC green fluorescence. Both in human (left panel) as well as in rat (right panel) DRG neurons, KOR and DOR immunoreactivity is clearly detectable and colocalizes (yellow fluorescence) abundantly with the sensory neuron marker CGRP (double arrow). Notably, KOR was also expressed in satellite glia cells (arrowhead) encircling KOR-positively or -negatively (*) stained DRG neuronal cell bodies. Bars represent 55 µm.

Figure 5

Detection of the opioid peptide precursors POMC, PENK, and PDYN in human versus rat sensory DRG neurons. POMC, PENK, and PDYN were visualized by specific antibodies (see Supplementary Table S3) and respective secondary antibodies labelled with FITC green fluorescence; because their neuronal cell staining was scarce, tissue sections were co-stained with the pan-neuronal marker PGP9.5 for all DRG neurons, visualized by Texas red fluorescence; since PDYN immunostaining was even less abundant, sections were counterstained only with the nuclear stain 4′-6-Diamidino-2-phenylindole (DAPI) for better visibility. The pictures clearly show that POMC or PENK immunoreactivity was exclusively visible in PGP9.5-positive DRG neurons (colocalisation, yellow fluorescence); also, notably, many PGP9.5 immunoreactive neurons lacked POMC or PENK immunoreactivity. Bars represent 55 µm.

Figure 6

Detection of opioid receptor and opioid peptide precursor mRNA transcripts in human (A,B) versus rat (C,D) sensory DRG neurons. In using specific mRNA primers, quantitative real-time PCR showed a predominant mRNA expression of MOR over DOR and KOR in human DRG (5-fold), as well as MOR over DOR and KOR in rat (10-fold) sensory DRG neurons (A,C) (p < 0.05, Kruskal–Wallis test, followed by post hoc Dunn’s test). In addition, quantitative real-time PCR analyses of human DRG neurons revealed that PENK opioid peptide precursor mRNA was more abundant than POMC and PDYN mRNA, whereas POMC mRNA was superior in rat DRG neurons (B,D). Data are expressed as means ± SD. Statistical significance was calculated using the Kruskal–Wallis test, followed by post hoc Tukey’s test, with * p < 0.05.

Figure 7

Detection of MOR mRNA transcripts in relation to key pain-signaling molecules in human (A) versus rat (B) sensory DRG neurons. In using specific mRNA primers, quantitative real-time PCR showed the expression of key pain-signaling-molecule mRNAs in relation to MOR mRNA transcripts of both human (A) and rat (B) sensory DRG neurons. Moreover, quantitative real-time PCR analyses of human DRG neurons revealed that Piezo2 mRNA was more abundant than that of other key pain-signaling molecules, whereas TRPA1 mRNA was superior in rat DRG neurons (A). Data are expressed as means ± SD. ΔCt values were obtained by Ct_gene–Ct_18S housekeeping gene and subsequently related to ΔCt values of MOR according to the ΔΔCt method [24].

Figure 8

Inhibition of voltage-dependent calcium currents by the MOR agonist fentanyl in rat DRG neurons. Whole-cell patch-clamp recordings in isolated rat DRG neurons were used to assess fentanyl’s dose-dependent inhibition of +10 mV-evoked inward calcium currents (A). This inhibition was reversed in a dose-dependent manner by the MOR antagonist naloxone (B). The most effective fentanyl dose reduced calcium currents by approximately 40%, and naloxone completely abolished this effect, confirming MOR specificity on VDCCs (C). *** p < 0.001 comparison of the most effective fentanyl-induced inhibition vs. fentanyl + naloxone-treated DRG neurons (n = 16–20); two-tailed t-test. Data are presented as means ± SEM.