Two selective δ agonists were used throughout the study: SNC80 (Calderon et al., 1994
), and ARM390 (Wei et al., 2000
). Both compounds show similar receptor binding and G-protein activation properties (Wei et al., 2000
; Marie et al., 2003
; Pradhan et al., 2009
), and produce comparable pain-relieving effects in models of inflammatory pain (Pradhan et al., 2009
). The two compounds penetrate the blood brain barrier following systemic injection and produce centrally mediated emotional responses (our unpublished results). However, in both cell systems and in vivo
, SNC80 triggers robust δ receptor internalization, whereas ARM390 does not produce significant receptor trafficking (Marie et al., 2003
; Pradhan et al., 2009
Acute SNC80, but not ARM390 treatment produces transient desensitization of the analgesic response
We previously showed that SNC80, but not ARM390,produced acute behavioral desensitization 4 h after acute administration; and that this response was restored 24 h after the initial injection (Pradhan et al., 2009
). Here we first examined whether acute behavioral desensitization could be observed at a later time point (12 h) and also confirmed the recovery of receptor function after 24 h. CFA was injected into the tail of DOR-eGFP mice, and strong heat hyperalgesia was observed 48 h later (, controls vs dashed lines). An initial injection of either SNC80 or ARM390 reversed the CFA-induced hyperalgesia, and the two compounds were equally effective (, Injection 1). Twelve hours after the first injection, a second injection of SNC80 was ineffective, while 24 h after the first injection the antihyperalgesic effect of the compound was restored (, Injection 2, 12 H and 24 H). ARM390 produced potent antihyperalgesia at the second injection, both 12 and 24 h after the first injection (, Injection 2, 12 H and 24 H). A parallel group of DOR-eGFP animals were killed at the time that they would have received the second drug injection. We observed robust DOR-eGFP internalization 12 h after SNC80 (, 12 H), and cell surface fluorescence was restored at the 24 h time point (, 24 H). In contrast, ARM390 treatment did not induce receptor internalization at any time examined (, 12 H and 24 H).
Figure 1 Acute SNC80, but not ARM390, produces transient receptor internalization and behavioral desensitization in DOR-eGFP mice. A, Desensitization (12 H) and recovery (24 H) of the analgesic response in the CFA tail model of inflammatory pain. Injection 1, (more ...)
Thus, as we showed previously (Pradhan et al., 2009
), receptor internalization (SNC80 but not ARM390) leads to acute desensitization of the analgesic response, observable 4 H and 12 H after treatment. Importantly, 24 h following treatment with either SNC80 or ARM390, DOR-eGFP was detected on the cell surface and these receptors were fully functional, regardless of whether receptor internalization had occurred previously or not.
Chronic SNC80 and ARM390 treatments produce full analgesic tolerance, independent of receptor internalization
Because SNC80- and ARM390-exposed animals appeared indistinguishable 24 h after the first treatment, we examined the consequences of chronic SNC80 or ARM390 treatment every 24 h. We first tested heat hyperalgesia in DOR-eGFP mice (, left). Animals were injected with CFA into the tail, and 48 h later were treated with vehicle, SNC80 (10 mg/kg) or ARM390 (10 mg/kg) every 24 h for 5 d. Heat hyperalgesia was stably expressed over the 5 d, as observed in control animals. As expected, both SNC80 and ARM390 efficiently reduced heat hyperalgesia on day 1. The drug effect gradually decreased over the 5 d, regardless of the internalization properties of the compounds. The time course for the development of tolerance was comparable for the two drugs, and complete tolerance was observed at day 5. We repeated the same experiment using wild type C57BL/6J mice (, middle). Tolerance developed similarly for the two δ agonists indicating that this observation was not specific to the DOR-eGFP strain. Finally, we determined whether tolerance to SNC80 and ARM390 was endpoint specific. We injected wild type mice with CFA in the paw, and tested mechanical allodynia by von Frey hair stimulation after 48 h (, right). Again, tolerance was observed in both SNC80- and ARM390-treated animals. As with heat hyperalgesia, tolerance developed to the two drugs at the same rate, and was complete by day 5. Together, our results show that analgesic tolerance develops for both SNC80 and ARM390, independent of the high- or low-internalizing properties of the agonist.
Figure 2 Chronic SNC80 andARM390both produce analgesic tolerance. Development of tolerance in two mouse strains and two pain models. All animals were tested every 24 h with vehicle (control), SNC80 (10 mg/kg), or ARM390 (10 mg/kg) for 5 d. Left, CFA tail, thermal (more ...)
SNC80- and ARM390-tolerant animals show distinct receptor modifications
We then investigated the status of δ-opioid receptors after the establishment of tolerance to either SNC80 or ARM390. We examined DOR-eGFP receptor subcellular localization, ligand binding and G-protein coupling. As in , DOR-eGFP mice were subjected to CFA injection in the tail, and treated for 5 d with vehicle, SNC80 or ARM390. Full tolerance was observed by day 5 (data not shown), and animals were killed 24 h after the last injection (day 6) for ex vivo
analysis. SNC80-tolerant animals showed cell surface expression of DOR-eGFP in all areas examined (). Importantly however, the fluorescent signal was significantly reduced, suggesting that receptor downregulation had occurred. Further, [3
H]naltrindole binding was undetectable in brain membranes (), and [35
S]GTPγS binding was severely attenuated in the brain () and spinal cord (). These results indicate that repeated SNC80 treatment leads to strong receptor downregulation throughout the nervous system. This result is consistent with our previous observation of lysosomal targeting of DOR-eGFP following agonist-induced receptor internalization (Pradhan et al., 2009
, see references therein).
Figure 3 Chronic SNC80 and ARM390 produce distinct adaptive responses at the receptor level. A, Subcellular DOR-eGFP localization. Brain and dorsal root ganglia were analyzed 24 h after the last drug treatment (day 6) by confocal microscopy, and representative (more ...)
In sharp contrast, ARM390-tolerant animals showed intense DOR-eGFP surface fluorescence in both DRG and hippocampus, comparable to the control group (). Further, ARM390-tolerant animals showed [3H]naltrindole () and [35S]GTPγS binding in brain (), and spinal cord () membrane preparations that were comparable to the control group. Hence, δ-opioid receptor surface expression, receptor number and receptor coupling to G-proteins are intact in ARM390-tolerant animals. In contrast to SNC80, tolerance to ARM390 does not seem to involve adaptive mechanisms at the receptor level.
SNC80- and ARM390-tolerant animals show cross-tolerance to analgesic responses
At this stage of the study, the finding that δ-opioid receptors were intact and expressed on the cell surface in ARM390-tolerant animals, was most intriguing. This observation led us to examine analgesic responses of these animals to SNC80, using a cross-over design. A first experiment was performed in DOR-eGFP mice, using the CFA paw model (). As in the previous experiments, mice were made tolerant to SNC80 or ARM390 by daily treatments over 5 d. On the sixth day, SNC80-tolerant animals were challenged with ARM390, and ARM390-tolerant animals were challenged with SNC80. An antiallodynic effect was not detected for either compound, indicating that cross-tolerance had established in both SNC80- and ARM390-tolerant groups. We performed a second cross-tolerance experiment, using wild-type C57BL/6J mice in the CFA tail model (). Again, none of the compounds showed antihyperalgesic activity in tolerant animals, indicating that cross-tolerance developed independent of mouse strain or analgesic endpoint (mechanical or thermal).
Figure 4 Chronic SNC80 and ARM390 both produce analgesic cross-tolerance. SNC80-tolerant mice were challenged with ARM390 (10 mg/kg), and ARM390-tolerant mice were challenged with SNC80 (10mg/kg). Graphs show tolerance and cross-tolerance data. A, CFA paw, mechanical (more ...)
As ARM390 produced tolerance and cross-tolerance in the absence of changes at the receptor level, we explored the possibility that this tolerance was due to associative tolerance resulting from our repeated testing paradigm. Consistent with previous reports (Gamble and Milne, 1989
; Williams et al., 2001
, see references therein), repeatedly testing mice for 5 d with vehicle resulted in an attenuation of analgesic effects, and this was observed for both SNC80 and ARM390 (). However, this associative tolerance only partially contributed to the full tolerance produced by either δ agonist as both still produced a significant antiallodynic effect in the habituated group. These results indicate that the observed tolerance to either SNC80 or ARM390 resulted from both associative and nonassociative (drug) factors, and confirmed the development of drug-induced tolerance in response to chronic ARM390.
Altogether, these data show that neither SNC80-tolerant animals, whose receptors are downregulated, nor ARM390-tolerant animals, whose receptors are intact at the cellular level, respond to any δ agonist in a model of pain. We conclude that repeated agonist treatment abolishes δ-opioid analgesia, either via internalization-dependent or internalization-independent mechanisms.
ARM390-tolerant mice respond to SNC80 at receptor trafficking and behavioral levels
We further investigated δ-opioid receptor function in ARM390-tolerant animals. We first examined SNC80-induced receptor internalization in both SNC80- and ARM390-tolerant animals. As in previous experiments, mice were made tolerant to SNC80 or ARM390 over 5 d. On the sixth day SNC80-tolerant animals were challenged with ARM390, ARM390-tolerant animals were challenged with SNC80, and tissue from central and peripheral nervous systems were analyzed by confocal microscopy. In accordance with our previous observation (), SNC80-tolerant animals showed DOR-eGFP downregulation, as evidenced by significantly reduced fluorescence in the hippocampus, striatum and dorsal root ganglia (). Interestingly, robust DOR-eGFP internalization was observed in ARM390-tolerant animals upon challenge with SNC80, in both the central and peripheral nervous system (). In combination with receptor binding and G-protein coupling data from tolerant animals (), this result confirms that chronic treatment with ARM390 leaves δ receptors functionally intact at the cell surface and readily responsive to agonist-induced internalization.
Figure 5 Chronic ARM390 induces analgesic tolerance only, leaving other δ-opioid receptor responses intact. A, SNC80-induced internalization in SNC80- and ARM390-tolerant animals. Striatum, hippocampus, and dorsal root ganglia were analyzed by confocal (more ...)
Because SNC80 was able to internalize δ-opioid receptors in the ARM390-tolerant animals, but was unable to produce antiallodynic or antihyperalgesic effects (), we tested whether SNC80 could elicit other behavioral responses that do not involve pain processing mechanisms. SNC80 is known to produce locomotor activation (Spina et al., 1998
) and anxiolytic responses (Saitoh et al., 2004
) and we therefore tested both behaviors. We treated three groups of wild-type C57BL/6J mice with either vehicle (control), SNC80 (SNC80-tolerant) or ARM390 (ARM390-tolerant) and established tolerance over 5 d, as described previously. On day 6, mice in each group were challenged in a locomotor test with either vehicle or SNC80 (). SNC80-tolerant animals were unresponsive to the locomotor stimulant effect of SNC80, consistent with a previous report (Jutkiewicz et al., 2005
). In contrast, robust SNC80-induced locomotor stimulation was observed in both control and ARM390-tolerant animals. The anxiolytic effect of SNC80 was examined in another experimental group, and similar results were obtained (). Acute SNC80 challenge did not modify the behavior of SNC80-tolerant animals in the elevated plus maze. However, control and ARM390-tolerant animals did respond to acute SNC80 and spent significantly more time in the open arms, indicating a reduction in anxiety levels. Therefore, after a 5 d chronic regimen, ARM390 treatment produced tolerance and cross-tolerance at the level of pain responses, while agonist-induced locomotor activation and anxiolysis remained intact.
ARM390-tolerant animals show desensitization at the level of the dorsal root ganglia
We next investigated the mechanism of ARM390-induced analgesic tolerance. We focused on δ receptor activity at the level of the DRG, as these contain first order neurons in pain transmission, but are not primary mediators of locomotor and emotional processing. Due to technical limitations we could not analyze δ receptor radioligand or [35
S]GTPγS binding in the DRGs, as performed in brain and spinal cord membranes. A main downstream signaling effector of δ receptors are VDCCs, which are inhibited by δ agonists in DRGs (Walwyn et al., 2005
). As in previous experiments, wild-type C57BL/6 mice were treated with CFA in the paw, and made tolerant to SNC80 or ARM390 over 5 d. On day 6, we measured SNC80-induced Ca2+
channel inhibition in DRGs collected from these animals (). The SNC80 challenge was ineffective in DRGs from naive animals, as previously described (Walwyn et al., 2005
). In contrast, SNC80 efficiently inhibited Ca2+
channels following CFA-induced inflammation, consistent with enhanced δ receptor function following chronic pain (Cahill et al., 2003
). In DRGs from SNC80-tolerant animals, δ-mediated regulation of Ca2+
channel activity was significantly reduced, in agreement with δ receptor downregulation following chronic treatment with this drug (). Remarkably, δ-regulated Ca2+
channel inhibition was also reduced, and even abolished in DRGs from ARM390-tolerant mice. Therefore, chronic treatment with ARM390 does not change surface localization (), receptor number (), coupling to G-proteins in the CNS (), or ability to internalize (), but does desensitize δ receptor-VDCC coupling. This disruption of δ receptor-Ca2+
channel interaction in primary nociceptors could explain the analgesic tolerance in ARM390-tolerant animals.
Figure 6 Chronic SNC80 and ARM390 both reduce δ receptor-mediated inhibition of Ca2+ channels in DRGs. The inhibitory effect of SNC80 (1 µm) on voltage-dependent Ca2+ channels was measured in medium-large DRG neurons. A, B, Exemplar currents from (more ...)