Although much of what we know about the properties of nociceptive afferents was obtained from the study of rodent sensory neurons, a number of recent studies of human sensory neurons have shown that in addition to important similarities between rodent and human sensory neurons,11,28,30 there are marked differences as well7,34,41,52,72,73 ranging from gene expression12,41,52 to pharmacology and biophysical properties.12,71,72 The issue of species differences may be a particularly important question to address in the context of opioid receptors both because of the ongoing opioid crisis in the United States and because of several recent high-profile articles highlighting the importance of opioid receptors on primary afferent neurons to the analgesic action of these drugs as well as the development of tolerance. Evidence has accumulated that opioid receptor subtypes other than the μ-opioid peptide (MOP9) receptor (the primary target for morphine and related compounds), such as the δ-opioid peptide (DOP,1,49) receptor, κ-opioid peptide (KOP,33,60) receptor, and nociceptin/orphanin-FQ opioid peptide (NOP,10,19,23) receptor, may serve as analgesic targets devoid of the deleterious consequences associated with MOP receptor agonists. Indeed, large differences have been documented between rodents and primates in opioid expression profiles.6,10,24 Thus, the purpose of this study was to characterize the distribution of functional opioid receptor subtypes in human dorsal root ganglion (DRG) neurons.
The possibility that DOP, KOP, and/or NOP receptors may serve as alternative therapeutic targets for the treatment of pain is largely supported by rodent data demonstrating the analgesic efficacy of peripherally administered DOP,35 KOP,8,33,60 and NOP13,23,25 receptor agonists. Whether this reflects agonist activity on a subpopulation of afferents overlapping that expressing MOP receptors remains an area of controversy for DOP receptors with conflicting evidence of both a high level of receptor coexpression in small-diameter peptidergic DRG neurons21,40,53,56,61,70 and a pattern of expression that is largely nonoverlapping.47,48 Data from both rat and mouse are more consistent and suggest that although there is clearly an overlap in the expression of MOP and KOP receptors, KOP receptors are more widely distributed.21,29,54 By contrast, in situ analysis showed that NOP receptors are predominantly expressed in a nonoverlapping subpopulation of medium to large cell body diameter DRG neurons.37
Our results support the suggestion that DOP, KOP and NOP receptor agonists may be viable alternatives to MOP receptor agonists for the treatment of pain.
2. Materials and methods
2.1. Human dorsal root ganglion collection and culture
Lumbar 4 and 5 DRG from human donors were obtained with the consent of their next of kin to use their family member's tissue for research purposes. Collection and study of the DRG was approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents. Consent was obtained by Center for Organ Recovery and Education staff, as was the collection of tissue. Dorsal root ganglia were collected as previously described in Ref. 73 and immediately placed in ice-cold collection media (in mM: 124.5 NaCl, 5 KCl, 1.2 MgSO4, 1 CaCl2, and 30 HEPES) pH with NaOH to 7.2. Dorsal root ganglia were then cleaned of connective tissue, cut into small (∼1 mm3) pieces, and enzymatically treated with 6.25% (wt/vol) collagenase P (Sigma-Aldrich, St Louis, MO) for 1 to 2.5 hours at 37°C before mechanical dissociation. These final steps were initiated for all donors within 6 hours after cross-clamp (the time at which systemic circulation was stopped). Dissociation and plating protocols73 as well as solution components73 were followed as previously described. Neurons were studied between 12 hours and 5 days after plating.
2.2. Patch-clamp electrophysiology
Standard whole-cell patch clamp techniques were used to record voltage-gated Ca2+ currents (VGCCs) in human DRG neurons. Data were collected with HEKA EPC10 controlled by Patchmaster (v 2 × 90.5) software (HEKA Electronik GmbH, Lambrecht/Pfalz, Germany). Data were acquired at 5 to 20 kHz and filtered at 1 to 4 kHz. Borosilicate glass (WPI, Sarasota, FL) electrodes were 1 to 2 MΩ when filled with the electrode solution containing (in mM): 130 Choline-Cl, 3 KCl, 5 BaCl2, 0.6 MgCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.4 with 10% wt/vol tris-base, and the osmolarity was adjusted to 320 mosmol·kg−1 with sucrose. The electrode solution consisted of (in mM): 110 TEA-MES, 40 TEA-Cl, 2 MgCl2, 1 CaCl2, 11 EGTA, 10 HEPES, 2 Mg-ATP, and 1 Li-GTP. The pH was adjusted to 7.2 with 10% wt/vol tris-base, and the osmolarity was adjusted to 310 mosmol·kg−1 with sucrose.
2.3. Ca2+ imaging
Human DRG neurons were loaded with 2.5 μM Ca2+ indicator fura-2 AM ester with 0.025% wt/vol Pluronic F-127 in normal bath solution (NB) for 30 minutes at 37°C. Normal bath solution consisted of (in mM): 130 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.4 with 10% wt/vol tris-base, and the osmolarity was adjusted to 320 mosmol·kg−1 with sucrose. Neurons were then placed in a recording chamber continuously perfused with NB. Fluorescence data were acquired on a PC running MetaFluor software (Molecular Devices, Sunnyvale, CA) via an EMCCD camera (model QuantEM 512SC; Photometrics, Tucson, AZ). For Fura-2 imaging, the ratio (R) of fluorescence emission (510 nm) in response to 340/380 nm excitation (controlled by a DG-4 [Sutter Instrument, Novato, CA]) was acquired at 1 Hz during application of KCl (50 mM) or capsaicin (500 nM), which were applied through a computer-controlled, piezo-driven perfusion system (Fast-Step Model SF-77B; Warner Instruments, Hamden, CT). The advantage of this system is that with the cells in the field of view under the direct flow from the perfusion pipette perfused with a flow rate of approximately 1 mL/minutes, it is possible to completely exchange the solution around the cells very rapidly without having to exchange the entire recording chamber volume. The rate of exchange was confirmed with cell-free patch electrodes in which the holding current was measured after application and wash of a solution containing 50-mM KCl. The holding current change was complete within 20 ms and stable with each change of solution (data not shown). Neurons were considered responsive to KCl and/or capsaicin if their application was associated with an increase in fluorescence >2 SDs above baseline. Similarly, neurons were considered responsive to opioid receptor agonists if the KCl-evoked response was changed >2 SDs from the baseline KCl-evoked response. Data are plotted as a change in fluorescence (ΔF) over baseline fluorescence (F).
DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin), DADLE ([D-Ala2, D-Leu5]-enkephalin), dynorphin, nociceptin/orphanin-FQ, CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2), and naltrindole were dissolved in water. Nor-binaltorphimine (nor-BNI), bpV(Phen), and SB 612111 were dissolved in DMSO. Naloxone and (±)-J113397 were dissolved in ethanol. All test agents were dissolved as 100× to 1000× stocks and stored at −20°C until use. Opioid receptor agonists and antagonists were obtained from Sigma-Aldrich and/or Tocris Bioscience (Bristol, UK), whereas bpV(Phen) was obtained from Cayman Chemicals (Ann Arbor, MI).
2.5. Single-cell semi-quantitative reverse transcriptase polymerase chain reaction
Dissociated human DRG neurons were collected with large bore (∼40 µm) pipettes, expelled into tubes containing 3 µL of extraction buffer, and stored at −80°C until processed. Transcripts from single cells were reverse transcribed and linearly preamplified using the MessageBOOSTER kit for cell lysate (Epicentre). After preamplification, the products were cleaned with RNA Cleaner & Concentrator-5 columns (Zymo Research, Irvine, CA) and transcript levels were quantified using quantitative polymerase chain reaction (PCR) with optimized primers and SsoAdvanced SYBR Green Master Mix (BioRad, Hercules, CA). Cycle-time (Ct) values were determined using regression. Quantification threshold was determined to be interreplicate average of 32 Ct, the point where replicates have a 95% chance of reoccurring, and the GAPDH threshold for cell inclusion was set to 23 Ct to ensure we could detect transcripts a 1000-times less prevalent than GAPDH. Bacterial RNA for Lys was added to each mixture as an external control. Primer sequences are listed in Table 1.
2.6. Statistical analyses
In accordance with previous studies involving human DRG, no á priori analyses were performed to estimate the number of donors necessary to complete this study. The statistical tests used to compare groups and/or treatments are indicated in the figure legends. Unless otherwise specified, data are presented as mean ± SEM.
Over 1500 DRG neurons from 54 donors were studied for the characterization of opioid receptors. These donors consisted of 23 females and 31 males belonging to the age range between 18 and 77 years, with a median age of approximately 45 years. The majority of the donors were white with the exception of 3 African Americans, 1 Asian American, and 1 Native Hawaiian. A breakdown of the donors by sex, age, and race can be found in Table 2.
3.1. The contribution of voltage-gated Ca2+ channels to the actions of opioids and KCl-evoked Ca2+ transients in human dorsal root ganglion neurons
Rodent data suggest that the primary mechanism underlying the analgesic action of spinal opioids is the inhibition of transmitter release from primary afferents26,38 secondary to the inhibition of VGCC.22,63 The analgesic effects of inhibiting transmitter release from central terminals is in contrast to the inhibition of VGCC in peripheral terminals, which, while suppressing the efferent release of transmitter in the periphery, does not seem to influence nociceptor excitability and consequently nociceptive threshold.51 And although the efferent release of transmitter may contribute to the inflammatory response and consequently pain associated with tissue injury,64 this distinction helps explain why peripheral administration of opioid receptor agonists have been generally shown to have little analgesic effect in the absence of tissue injury.2,27,59 Thus, although other functional assays could be used to screen for the antihyperalgesic actions of opioid receptor agonists,15 the focus on VGCC inhibition in this study should be considered a screen for potential analgesic effects, particularly if agonists were administered spinally. To confirm that a similar mechanism occurs in human DRG neurons, we assessed the impact of the selective MOP receptor agonist, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO, 1 µM), on VGCC evoked in human DRG neurons with patch-clamp electrophysiology. DAMGO significantly reduced VGCC (Fig. 1A) by an average of 37.9 ± 5.9% (n = 10). There was a small decay in the magnitude of inhibition with a three-minute application of DAMGO, and although recovery from inhibition was relatively slow, it was largely complete after 3 minutes of washing (Fig. 1A, bottom). To at least begin to assess whether other opioid receptor agonists act through a comparable mechanism, we screened for the effects of the KOP agonist (dynorphin) and NOP agonist nociceptin on VGCC in human DRG neurons. Currents were inhibited by dynorphin in 2 of 3 neurons tested and nociceptin in 2 of 4 neurons tested (data not shown). Two mechanisms have been described underlying G-protein-coupled receptor-induced suppression of VGCC: a protein kinase C-dependent process that is relatively slow to develop and reverse, which is associated with a decrease in the number of functional channels in the membrane,57,67,68,74 and a membrane delimited rapid Gβγ-dependent mechanism, involving the displacement of the voltage-gated Ca2+ channel β subunit,18,65 and a depolarizing shift in the voltage dependence of channel activation. Because it is possible to reverse this second form of inhibition with a depolarizing voltage step, a process referred to as prepulse potentiation, we screened for the latter mechanism of VGCC inhibition with a prepulse protocol (Fig. 1B, top). Consistent with a Gβγ-dependent mechanism of VGCC inhibition, a prepulse to +80 mV partially reversed DAMGO-induced inhibition of VGCCs (Fig. 1B, bottom).
Because of the capability to screen multiple neurons at the same time for their responses to opioid receptor agonists with Ca2+ imaging, we next sought to confirm that KCl-evoked Ca2+ transients, as assessed with fura-2 microfluorimetry, was dependent on Ca2+ influx through VGCCs. After establishing a stable baseline response to KCl in NB (Fig. 1C), the bath solution was changed to a nominally Ca2+-free bath solution (0 Ca2+ but no Ca2+ chelator added). In the presence of Ca2+-free bath, the evoked Ca2+ transient was reduced by 95.7 ± 1.2% (Fig. 1D, n = 29). Moreover, when high- and low-threshold VGCC blockers, 100 μM CdCl2 and 100 μM NiSO4, respectively, were added to NB, the evoked Ca2+ transient was reduced (Fig. 1E) by 74.6 ± 1.7% (Fig. 1F, n = 9). Together, these data suggest that KCl-evoked Ca2+ transients can be used to screen for the presence of functional opioid receptors in human DRG neurons.
3.2. Distribution of functional μ-opioid peptide receptors among human dorsal root ganglion neurons
The distribution of MOP receptors among rodent sensory neurons has been well described functionally, where in mouse and rat DRG neurons, it seems to be primarily restricted to a subpopulation of small-diameter neurons also responsive to the algogen capsaicin.44,50,58,62 To assess the distribution of functional MOP receptors among human DRG neurons, we assessed the impact of DAMGO on KCl-evoked Ca2+ transients (Fig. 2A). Based on our criterion for considering a neuron responsive to an agonist (ie, a change in evoked transient >2 SDs from the baseline KCl response), 30 of 58 neurons tested (51.7%) were responsive to DAMGO. In these neurons, the average maximal suppression of the evoked transient was 42 ± 5% (Fig. 2B). In addition, 15 of these neurons were tested for capsaicin sensitivity and 12 were responsive, suggesting that the majority of the neurons are nociceptive.
The inhibitory actions of DAMGO were concentration dependent (Fig. 2B). The lowest concentration of DAMGO with which we were able to detect an effect was 30 nM. However, both the magnitude of the inhibition and the proportion of neurons responsive to DAMGO (Fig. 2B, inset) increased in response to concentrations of DAMGO higher than 30 nM. The EC50 for DAMGO was 56 nM. Consistent with the suggestion that DAMGO is a selective MOP receptor agonist, the MOP receptor antagonist naloxone (1 μM) (Fig. 2C, top) significantly attenuated the DAMGO-induced suppression of the evoked Ca2+ transients to 13.3 ± 1.2% (Fig. 2D, bottom), but did not affect the proportion of DAMGO responders (Fig. 2D, top). Curiously, the MOP receptor antagonist CTOP (10 μM) (Fig. 2C, bottom) alone produced an inhibition of 31.1 ± 4.0% (n = 11; data not shown) of the evoked Ca2+ transient. This effect was seen across 5 donors as well as different drug batches from different vendors. Further characterization revealed that the CTOP-induced inhibition of the evoked Ca2+ transient was not blocked by naloxone (n = 10, P = 0.89, paired t test; data not shown). CTOP attenuated the evoked Ca2+ transient with an EC50 of 0.55 μM with a maximal inhibition of 31% at 3 μM (data not shown). This inhibition was unlikely to be a solution exchange artifact either because there was no change in background fluorescence associated with the addition of CTOP containing solution. Nevertheless, CTOP (10 μM) decreased the proportion of DAMGO-responsive neurons (Fig. 2D, top), as well as the magnitude of the DAMGO-induced suppression of the evoked Ca2+ transient (to 20.1 ± 2.8%). Furthermore, when neurons were challenged with DAMGO again after wash of CTOP, there was a significant increase in the magnitude of the DAMGO-induced suppression of the evoked Ca2+ transient (Fig. 2D, bottom). In contrast to CTOP, naloxone alone had no detectable influence on either resting Ca2+ or KCl-evoked Ca2+ transients in the majority of neurons tested. However, in neurons isolated from one donor, a naloxone-induced increase in intracellular Ca2+ was detected (not shown). Curiously, this particular donor had a medical history of opioid abuse, and died of apparent overdose. These data are in line with evidence suggesting that the MOP receptor signaling is altered after exposure to MOP receptor agonists.17,32,69 We observed no age-dependent influence on the number of DAMGO responders (≥65 yo; n = 4 donors vs <30 yo; n = 3 donors). However, there was a significant influence of sex on the number of neurons responsive to DAMGO (females n = 8 donors; 44/97 [45.4%] vs males n = 7 donors; 99/153 [64.7%], **P = 0.003, χ2).
3.3. Distribution of functional δ-opioid peptide receptors among human dorsal root ganglion neurons
We next sought to assess the distribution of DOP receptor among human DRG neurons. δ-opioid peptide receptor signaling among DRG neurons remains one of the more controversial topics in the opioid literature. One line of evidence from both rat and mouse suggests that DOP receptors are present in a subpopulation of DRG neurons largely overlapping with that expressing MOP receptors.21,40,61 In these neurons, however, DOP receptors are not normally present on the plasma membrane, and are only trafficked to the membrane after high levels of activity43 and/or changes in the balance of phosphatase/kinase activity.53 By contrast, an opposing line of evidence from the mouse suggests that DOP receptors are present in a population of medium to large neurons distinct from that expressing MOP receptors, where it is present in the plasma membrane even at rest.47,48 To begin to assess the presence of DOP receptors in human DRG neurons, we started with the putative DOP receptor selective agonist, SNC80, in an experiment similar to that used with DAMGO, with the exception that after wash of SCN80, we could also determine whether the neuron was responsive to DAMGO (Fig. 3A). Similar to the bulk of rodent data, we found that under basal conditions very few (1 of 42 [2.4%]) neurons from one donor responded to SNC80 at 1 μM. At 3 and 10 μM, we observed a marked increase in the number of responsive neurons and fractional inhibition of the evoked Ca2+ transient (Fig. 3B and inset). Such a high proportion of SNC80 responders were consistent with previous data suggesting that SCN80 has off-target effects at concentrations not much higher than that needed to produce a maximal DOP receptor response.48 However, assuming 1 μM SNC80 is still selective for DOP receptors, we next assessed the impact of preincubating DRG neurons in bpV(Phen) (10 μM for 30 minutes at 37°C), a phosphatase and tensin (PTEN) homolog inhibitor. This was based on recent data suggesting that under basal conditions, the balance of PI3Kinase to PTEN activity is such that DOP receptors are retained in the Golgi, whereas an increase in receptor phosphorylation is sufficient to enable DOP receptor trafficking to the plasma membrane.53 Consistent with the rodent data, we found that PTEN inhibition significantly increased the proportion of neurons responsive to SNC80 (1 μM) (12/25 [48%]) compared to vehicle-treated neurons (2/15 [13.3%]) (Fig. 3C, top). This increase in the proportion of SNC80 responders did not seem to be associated with an increase in the magnitude of the response (Fig. 3C, bottom). Neurons tested with SNC80 were also tested with DAMGO (1 μM) and capsaicin (500 nM). The distributions of vehicle and bpV(Phen)-treated neurons responsive to each test agent are plotted (Fig. 3D). Of note, the majority of SNC80 responsive neurons were also responsive to DAMGO and capsaicin (Fig. 3D). To test our assumption about the selectivity of 1 μM SNC80 for DOP receptors, we assessed the impact of a selective DOP receptor antagonist, naltrindole39 (3 and 10 μM), on the response to SNC80 (Fig. 3E). Interestingly, like CTOP, naltrindole alone at 10 μM also produced a small but significant (16.2 ± 1.6%) reduction in the evoked transient. Regardless of this effect, naltrindole at 3 and 10 μM had no detectable impact on the proportion of bpV(Phen)-treated neurons responsive to SNC80 (Fig. 3F, top). However, naltrindole at 10 μM decreased the magnitude of SNC80-induced inhibition of the evoked transient (Fig. 3F, bottom). These results suggest that naltrindole is not as potent against human DOP receptors in their native environment as it is in the rodent or heterologous expression systems. Age differences were not determined due to the lack of age range of donors screened with SNC80 (no donors younger than 30 years were screened). There was, however, the suggestion of a sex difference detected in the number of neurons responsive to SNC80 (females n = 2 donors; 9/25 [36%] vs males n = 3 donors; 6/56 [10.7%, **P = 0.007, χ2).
To further explore the distribution of DOP receptors among human DRG neurons, we assessed the impact of a second DOP receptor agonist, DADLE (Fig. 4A). In contrast to SNC80, we saw no effect of bPV(Phen) on the proportion of neurons that were responsive to DADLE (1 μM, bPV(Phen)- 18/40 [45%] vs vehicle- 9/26 [34.6%] Fig. 4B, top). Pretreatment with bpV(Phen) had no detectable influence on the magnitude of the DADLE-induced decrease in the evoked Ca2+ transient (Fig. 4B, bottom). As with SNC80, neurons tested with DADLE were also tested with DAMGO (1 μM) and capsaicin (500 nM). The distributions of vehicle and bpV(Phen)-treated neurons responsive to each test agent are plotted (Fig. 4C). Of note, the majority of DADLE responsive neurons were also responsive to DAMGO, and at least half were responsive to capsaicin. In addition, we repeated the naltrindole experiment with DADLE (Fig. 4D). This time, we used naltrindole at concentrations ranging from 1 to 10 µM. Regardless of antagonist concentration, there was no significant influence of naltrindole on the proportion of DADLE responders (Fig. 4E, top). However, the magnitude of the DADLE-induced suppression of the evoked Ca2+ transient was significantly attenuated by both 3-μM (data not shown) and 10-μM naltrindole (Fig. 4E, bottom). Age and sex differences were not determined due to the lack of age range (only 1 donor was aged younger than 30 years) and sex across donors screened with DADLE (only 1 male was screened).
3.4. Distribution of functional κ-opioid peptide receptors among human dorsal root ganglion neurons
Dynorphin was used as a KOP receptor agonist to assess the distribution of functional KOP receptors among human DRG neurons, as was described for MOP and DOP receptors (Fig. 5A). The effects of dynorphin were concentration dependent with both the proportion of dynorphin responders and the magnitude of the response increasing between 30 nM and 1 μM (Fig. 5B and inset). The EC50 for dynorphin-induced suppression of the evoked Ca2+ transient was 297 nM. Interestingly, although the majority of neurons responsive to dynorphin (1 μM) were also responsive to DAMGO, less than half were responsive to capsaicin (Fig. 5C). Finally, to confirm the involvement of KOP receptors in the actions of dynorphin, we used the KOP receptor selective antagonist nor-BNI (Fig. 4D). Although nor-BNI had no detectable influence on the resting or evoked Ca2+ transient, there was a significant reduction in both the proportion of dynorphin responders (Fig. 5E, top) and the magnitude of the dynorphin-induced suppression of the evoked Ca2+ transient (Fig. 5E, bottom). Age and sex differences were not determined due to the lack of age range (all donors were aged between 35 and 64 years) and sex across donors screened with dynorphin (only 1 male was screened).
3.5. Distribution of functional nociceptin/orphanin-FQ-opioid receptors among human dorsal root ganglion neurons
Finally, we assessed the distribution of functional NOP receptors among human DRG neurons as with the other opioid receptor subtypes (Fig. 6A). Nociceptin was used as the NOP receptor selective agonist, due to its low affinity for other opioid receptor subtypes.42 As with the other opioid receptor agonists, the effects of nociceptin were concentration dependent, with both the proportion of NOP receptor agonist responders and the magnitude of the effect increasing with nociceptin concentrations between 30 nM and 1 μM (Fig. 6B and inset). The EC50 for the nociceptin-induced suppression of the evoked Ca2+ transient was 75 nM. A subpopulation of neurons was also tested for sensitivity to nociceptin, DAMGO, and capsaicin. Although it was not clear whether it was a reflection of donor variability or some other factor, the proportion of neurons responsive to nociceptin when also tested for sensitivity to DAMGO and capsaicin was higher (∼65%, P = 0.04, chi-square test) than the proportion of neurons responsive to nociceptin, when the response to this ligand was tested alone (∼40%), regardless of whether the neuron was first challenged with nociceptin or DAMGO. Regardless, more than half of the nociceptin-responsive neurons were also responsive to DAMGO, whereas a small majority of neurons were responsive to capsaicin (Fig. 6C).
Similar to the analyses of putatively selective MOP, DOP, and KOP receptor agonists, we assessed the impact of NOP receptor antagonists on the actions of nociceptin. We started with J113397 (10 µM, Figs. 6D and E), and found that J113397 alone inhibited the evoked Ca2+ transient (Fig. 6E) in 18/38 neurons with an average inhibition of 22.7 ± 2.8% (data not shown). Nevertheless, in the presence of the antagonist, only a small proportion of the neurons tested (5/36) were responsive to nociceptin (1 μM, Fig. 6D). Furthermore, after wash of the antagonist, the magnitude of the nociceptin-induced suppression of the evoked Ca2+ transient was larger than in the presence of this antagonist (Fig. 6F). Because of the effect of J113397 alone on the evoked Ca2+ transient, we tried a second NOP antagonist, SB 612111 (3 μM, Fig. 6G). Unlike J113397, SB 612111 alone did not inhibit evoked Ca2+ transients and had no influence on the proportion of nociceptin responsive neurons (Fig. 6D). However, the magnitude of the nociceptin-induced suppression of the evoked Ca2+ transient was significantly attenuated by SB 612111 (22.0% vs 28.4%, P = 0.0094, n = 16). Furthermore, when neurons were challenged with nociceptin again after wash of SB 612111, there was a significant increase in the magnitude of the nociceptin-induced suppression of the evoked Ca2+ transient (Fig. 6H). There was the suggestion of an influence of age on the number of nociceptin responders (≥65 yo n = 2 donors; 37/52 (71.1%) neurons vs <30 yo n = 2 donors; 14/40 (35%) neurons, ***P = 0.0005, χ2). Sex differences were not determined due to the lack of sex across multiple donors screened with nociceptin (only 1 female was screened).
3.6. Expression of opioid receptor subtypes among human dorsal root ganglion neurons
As a complimentary approach to the analysis of the distribution of functional opioid receptor subtypes among human DRG neurons, we performed a single-cell PCR analysis of opioid receptor subtypes among neurons from 5 donors (Fig. 7A). Donors consisted of 3 males and 2 females in the age range 22 to 70 years (Fig. 7B), of which 4 donors were white/Caucasian and the fifth was African American. We found on average OPRM1 was expressed in 24.0 ± 3.7%; OPRD1 in 4.5 ± 1.3%; OPRK1 in 49.7 ± 7.5%; OPRL1 in 48.4 ± 6.1%; and TRPV1 in 96.5 ± 1.6% of neurons.
The purpose of this study was to determine the distribution of functional opioid receptor subtypes among human DRG neurons. We found that activation of human DRG MOP receptors results in the inhibition of VGCCs. Consistent with this mode of action, opioid receptor agonists suppressed the Ca2+ transient evoked by a brief application of 50 mM K+. Agonist-induced suppression of evoked Ca2+ transients were concentration dependent at least for MOP, KOP, and NOP receptor agonists, and the effects of each receptor subtype selective agonist were attenuated by receptor subtype-specific antagonists. The pharmacology of DOP receptors was more complex. Two DOP receptor agonists were used, which seemed to have different pharmacological properties: pretreatment with bpV(Phen) increased the number of responders to SNC80 but the number of responders to DADLE was as high in untreated neurons as that responsive to SNC80 after bpV(Phen) treatment. However, the impact of both DOP receptor agonists was antagonized by the DOP receptor selective antagonist naltrindole. Overlap of functional MOP receptor with the other opioid receptor subtypes was relatively high for DOP receptors as well as for KOP and NOP receptors. The proportion of neurons expressing KOP and NOP receptors as detected with single-cell quantitative PCR was generally consistent with functional data. By contrast, MOP receptor mRNA was detected in ∼half the number of neurons in which a response to DAMGO was detected, and the disconnect was even larger for DOP receptors. Consistent with previous studies of human DRG neurons, this analysis of functional opioid receptors revealed similarities and potentially important species differences with respect to the distribution and pharmacology of these important receptors.
There were clearly similarities between our results and previous data from the study of rodent sensory neurons. Our results are largely consistent with evidence indicating that the analgesic effect of opioids is due to the inhibition of VGCC on primary afferents. The pharmacology of MOP, KOP, and NOP receptors in human DRG neurons are roughly comparable to that previously reported in rodents. The distribution of MOP and KOP receptor subtypes among human DRG neurons also seems to be comparable to that in rodents.47,54 Finally, the distribution of NOP receptors among human DRG neurons is comparable to that previously described in rats (and humans) where the receptor is present in both TRPV1+ and TRPV1- neurons.3
The most notable difference between human DRG neurons and previous data from rodents was the significant attenuation of the evoked Ca2+ transient by MOP, DOP, and NOP receptor selective antagonists CTOP, naltrindole, and J113397.66 Furthermore, despite evidence suggesting that the antagonists used are both potent and relatively selective, they were not nearly as effective as predicted. This was true regardless of whether the antagonist inhibited the evoked Ca2+ transient underscoring the potential importance of native environment on receptor pharmacology. A potentially related observation was that the combination of Cd2+ and Ni2+ did not block as much of the evoked Ca2+ transient as would have been predicted from rodent data.36 This suggests that either high K+ activates a VGCC-independent Ca2+ influx pathway, or like the opioid receptors studied here, and our previous data with voltage-gated Na+ currents,72 nicotinic acetyl choline receptors,71 and GABAA receptors73 in human DRG neurons, the pharmacology of VGCCs in human DRG neurons is different from that observed in rodent neurons or heterologous expression systems.
Similarly, although the naltrindole data are consistent with the suggestion that both SNC80 (1 μM) and DADLE (1 μM) effects were DOP receptor mediated, the observation that PTEN inhibition increased the number of SNC80 but not DADLE responders suggests that bpV(Phen) modified the signaling pathway activated by SNC8016 rather than releasing DOP receptors from Golgi.53 Consistent with this suggestion, there is evidence of ligand-directed signaling differences between rodent and human opioid receptors (albeit for KOP receptors45,46), as well as evidence that receptor phosphorylation influences whether agonists activate G-protein or β-arrestin pathways. Thus, it is possible that bpV(Phen) enables SNC80 to activate a G-protein-dependent pathway that is normally engaged by DADLE activation of DOP receptors in human DRG neurons. Such a mechanism could help explain the limited clinical success of DOP receptor selective agonists,55 particularly if the compounds tested have a pharmacological profile similar to that observed for SNC80. Regardless of why PTEN inhibition is needed for SNC80 but not DADLE activity, the presence of functional DOP receptors in ∼50% of human DRG neurons largely overlapping that with MOP receptors is consistent with older rat21 and mouse62 data, as well as more recent mouse data.31,53,61 Such a distribution is opposed to that described in other mouse studies suggesting that MOP and DOP receptors are in nonoverlapping subpopulations of DRG neurons,47,48 and mediate modality-specific analgesia. The low level of DOP receptor mRNA detected in human DRG neurons is also consistent with the former lines of evidence, where receptor activation seems to be necessary to drive an increase in DOP receptor mRNA,43 but not the latter (where DOP receptors seem to be constitutively expressed at high levels). One potential explanation for the differences in these data sets is that the majority of data supporting the nonoverlapping constitutive pattern of DOP receptor expression were based on a mouse line in which the OPRD1 had been modified to generate a GFP-tagged DOP receptor4,47,48; this mutation may not only impact receptor trafficking, but could also influence the pattern of expression.
There were at least 2 results from our single-cell PCR analysis that are worth noting. The first is the apparent disconnect between the pattern of expression for MOP, DOP, and TRPV1, and the distribution of functional receptors. This was most notable for DOP receptors at one extreme and TRPV1 at the other, but was also true for MOP receptors. That is, as noted, we obtained evidence of functional DOP receptors in ∼50% of DRG neurons, whereas the mRNA was detected in 5%. By contrast, capsaicin-evoked Ca2+ transients were only observed in 54% (421/775) of neurons tested, whereas TRPV1 mRNA was detected in 96% of neurons. As suggested above, the pattern of OPRD1 expression is similar to that reported in rats21 and consistent with activity-dependent regulation of expression.43 The relatively low level of OPRD1 expression observed at the single-cell level is also consistent with the relatively low level of expression previously reported with RNAseq analysis of whole DRG.12 By contrast, the pattern of TRPV1 expression is consistent, at least in part with an injury-induced upregulation of TRPV1 associated with putting the neurons in culture, given evidence from rat suggesting that nerve injury results in an increase in TRPV1 expression in subpopulations of neurons that do not normally express this receptor.5,14,20 This highlights a caveat of this study, which is that it was not only performed on tissue obtained after death, but after frank injury to the neurons studied. Nevertheless, the patterns of opioid receptor gene expression at the whole ganglia level is at least consistent with our single-cell data and argues against injury-induced changes in the patterns observed.41 Minimally, however, these data highlight the caution that should be employed when interpreting gene expression data, particularly with the level of sensitivity now possible with RNAseq-based approaches.
The second interesting observation was associated with the donor-to-donor variability. Although the majority of the donors passed away from stroke or cardiac arrest, a few donors had a history of opioid abuse including a donor with low OPRM1 expression (Fig. 7B). In addition to the variability between donors, there are other potential explanations for the differences between previously described opioid receptor distribution in rodents and the current study in human DRG. First, the majority of rodent studies were collected in males of similar age with limited genetic variability, whereas our study consists of approximately 43% females (Table 2). Second, recovery of tissue from experimental animals is relatively time-locked, whereas there was considerably more variability in the timing around the recovery and processing of tissue from organ donors. Third, rodents have relatively limited life experiences, whereas this is certainly not the case for donors (Table 2).
Our current study is the first to assess the distribution of functional opioid receptor subtypes in human DRG neurons. Our data highlight both similarities and potentially important differences between humans and rodents both with respect to the distribution of opioid receptor subtypes, and the impact of the reagents routinely used to study these receptors. The similarities noted validate the continued use of rodents for the study of opioid receptor pharmacology, at least receptors not genetically modified. However, the differences, in particular those related to receptor pharmacology, suggest that an analysis of the effects of opioid compounds currently approved for clinical use may shed some light on differences in clinical utility. The differences also suggest that it will be important, if not essential, to screen novel pharmacological compounds against human receptors in their native environment before initiating clinical trials.
Conflict of interest statement
The authors have no conflicts of interest to declare.
M.S. Gold has been a consultant to Grünenthal GmbH, Germany. T. Christoph and W. Schröder are employees of Grünenthal GmbH, Germany.
Supplemental video content
A video abstract associated with this article can be found at http://links.lww.com/PAIN/A971.
This work was supported by Grünenthal GmbH. It was also supported by grants from the National Institutes of Health F32NS110148 (J.K.M.), F32NS103231 (J.E.H.), T32NS073548 (J.E.H. and J.K.M.), R01NS096705 (H.R.K.), and R01NS083347 and R01DK107966 (M.S.G.). The authors acknowledge the Center for Organ Recovery and Education (CORE) for their help in coordinating receipt of tissue from organ donors. The authors thank the donor family members for graciously consenting to the use of their loved one's tissue for research purposes.
Author contributions: J.K. Moy contributed to the experimental design, collected and analyzed data, and wrote the first draft of the manuscript. J.E. Hartung, M.G. Duque, and R. Friedman collected and analyzed data. V. Nagarajan and E. Loeza-Alcocer contributed to the collection and analysis of data. H.R. Koerber, T. Christoph, and W. Schröder contributed to the experimental design and analysis of data. M.S. Gold contributed to the experimental design, data collection and analysis, and the preparation of the manuscript.
Data and materials availability: Yes.
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