The function of opioid receptors (DOR) expressed by peripheral pain-sensing neurons (nociceptors) is regulated by both cyclooxygenase- and lipoxygenase (LOX)-dependent arachidonic acid (AA) metabolites. Whereas cyclooxygenase metabolites enhance responsiveness, LOX metabolites elicit a refractory, nonsignaling state of the DOR receptor system for antinociceptive signaling. In this study, using high-performance liquid chromatography-tandem mass spectrometry analyses, we have found that the 12-/15-LOX metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, were elevated after treatment of adult rat primary sensory neuron cultures with AA. Exogenously applied 12-HETE and 15-HETE, but not 5-HETE, completely prevented DOR and opioid receptor (KOR) agonist-mediated inhibition of prostaglandin E2 (PGE)-stimulated cAMP accumulation, but not inhibition, by the 5-HT receptor agonist 5-carboxamidotryptamine in cultured peripheral sensory neurons and in Chinese hamster ovary (CHO) cells heterologously expressing DOR or KOR. Similarly, intraplantar injection of 12- or 15-HETE, either alone or in combination, prevented DOR agonist-mediated inhibition of PGE-evoked thermal allodynia. Further, both AA- and carrageenan-mediated induction of the nonresponsive state of the DOR system was blocked by an intraplantar coinjection of the 12-/15-LOX inhibitors baicalein and luteolin. In contrast to the regulation of cAMP signaling, pretreatment with 12- and 15-HETE had no effect on either DOR or KOR agonist- mediated activation of extracellular signal-regulated kinase in peripheral sensory neurons or CHO cells. These results suggest that the analgesic efficacy of peripherally restricted opioids for treatment of inflammatory pain may be enhanced by adjunct inhibition of LOX activity.

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Receptor heteromers often display distinct pharmacological and functional properties compared to the individual receptor constituents. In this study, we compared the properties of the DOP-KOP heteromer agonist, 6'-guanidinonaltrindole (6'-GNTI), with agonists for DOP ([D-Pen2,5]enkephalin [DPDPE]) and KOP (U50488) in peripheral sensory neurons in culture and in vivo. In primary cultures, all three agonists inhibited PGE 2 -stimulated cAMP accumulation as well as activated extracellular signal-regulated kinase ½ (ERK) with similar efficacy. ERK activation by U50488 was Gi-protein mediated but that by DPDPE or 6'-GNTI was Gi-protein independent (i.e., pertussis toxin insensitive). Brief pretreatment with DPDPE or U50488 resulted in loss of cAMP signaling, however, no desensitization occurred with 6'-GNTI pretreatment. In vivo, following intraplantar injection, all three agonists reduced thermal nociception. The dose-response curves for DPDPE and 6'-GNTI were monotonic whereas the curve for U50488 was an inverted U-shape. Inhibition of ERK blocked the downward phase and shifted the curve for U50488 to the right. Following intraplantar injection of carrageenan, antinociceptive responses to either DPDPE or U50488 were transient but could be prolonged with inhibitors of 12/15-lipoxgenases (LOX). By contrast, responsiveness to 6'-GNTI remained for a prolonged time in the absence of LOX inhibitors. Further, pretreatment with the 12/15-LOX metabolites, 12-and 15hydroxyeicosatetraenoic acid, abolished responses to U50488 and DPDPE but had no effect on 6'-GNTI-mediated responses either in cultures or in vivo. Overall, these results suggest that DOP-KOP heteromers exhibit unique signaling and functional regulation in peripheral sensory neurons and may be a promising therapeutic target for the treatment of pain.

Peripherally‐expressed kappa opioid receptors (KOR) are a promising target to treat inflammatory pain that would be devoid of CNS related adverse effects. We have found that local injectionof KOR agonists into the rat hindpaw produces a profound antinociceptive response that is mediated by activation of G protein‐coupled inwardly‐rectifying potassium (GIRK) channels. We confirmed with immunohistochemistry that both KOR and GIRK2 channels are expressed in intraepidermal nerve fibers (IENFS), which propagate nociceptive signals from the periphery to the spinal cord. Interestingly, we also found that KOR and GIRK2 channels are expressed in keratinocytes. Keratinocytes make up the majority of cells in the epidermis and evidence suggests that they play a role in pain and sensory processing. A variety of nociceptive signaling receptors are expressed in keratinocytes, including transient receptor potential vanilloid channels 1 through 4 (TRPV1‐4), suggesting that keratinocytes can modulate and possibly initiate nociception in the epidermis. Here we sought to determine if expression of GIRK channels in keratinocytes plays a role in KOR‐mediated peripheral antinociception. We assessed the effect of local hindpaw administration of a GIRK2 shRNA expression plasmid fused to a HIV‐1 Tat peptide that facilitates transport into cells. Unlike IENFs, keratinocytes are capable of transcription, thus we used this method to ensure knockdown would occur in keratinocytes and not in IENFs. Rats were injected intraplantarly (i.pl., 50ul) with either GIRK2 shRNA or scrambled non‐effective (sNE) shRNA plasmid complex along with Fugene HD daily for two days. 48 hours after the second injection, the rats were injected i.pl. with ML297 (33ug), a direct GIRK channel activator, and paw withdrawal latencies (PWL) to a thermal stimulus were determined over a 20 min period. ML297 produced a robust (+5 sec above baseline) antinociceptive response in contralateral (non‐shRNA plasmid injected) paws and in hindpaws injected with the sNE shRNA plasmid. Theantinociceptive response to ML297 was lost completely in hindpaws injected with GIRK2 shRNA plasmid. We next determined if local knockdown of GIRK2 in keratinocytes altered the behavioral response to the KOR agonist, U50,488. Following administration of either GIRK2 shRNA or sNE shRNA plasmid, rats were injected with PGE2 (i.pl., 0.3ug) with or without U50,488 (0.1ug) and PWL to the heat stimulus was measured. The sNE shRNA plasmid did not alter the KOR agonist‐mediated reduction of PGE2‐evoked thermal allodynia, however, the GIRK2 shRNA completely blocked the KOR‐mediated response. PGE2‐evoked thermal allodynia in the absence of agonist was unaltered in hindpaws treated with either shRNA plasmid. These data suggest that GIRK channels expressed in keratinocytes play an essential role in peripheral KOR‐mediated antinociceptive signaling in the rat hindpaw. Support or Funding Information This work is supported by NIH/NIDA RO1 DA 038645.

Targeting peripherally expressed kappa opioid receptors (KOR) is a promising approach to treat inflammatory pain that would be devoid of CNS related adverse effects. KOR are known to couple to Gi‐protein‐mediated signaling pathways involved in the suppression of neuronal firing and nociception. In this study, we sought to determine the involvement of Gi protein coupled inward rectifying potassium (GIRK) channels in peripheral KOR‐mediated antinociceptive behavior in rats. Rats were injected intraplantarly (i.pl., 50ul) with PGE2 (0.3ug) with or without the KOR agonist, U50488 (0.1ug) and paw withdrawal latencies (PWL) to a thermal stimulus were determined over a 20 min period. Treatment with U50488 completely reduced the thermal allodynia produced by PGE2. Consistent with activation of Gi proteins by KOR, we found that the reduction of PGE2‐evoked thermal allodynia by U50488 was lost completely 24h after i.pl. injection of pertussis toxin. To determine if GIRK channels mediated the antinociceptive response to KOR, we measured the effect of U50488 on PGE2‐evoked thermal allodynia 15 minutes after i.pl. injection of either vehicle or tertiapin Q (TPNQ), an inhibitor that blocks channels containing GIRK1 and GIRK2 subunits. Administration of TPNQ had no effect on either baseline PWL or PGE2‐evoked thermal allodynia, however, U50488‐mediated reduction of PGE2‐evoked thermal allodynia was lost after TPNQ administration. We next tested if direct activation of GIRK channels by i.pl. injection of the GIRK activator, ML297, could produce antinociceptive effects. ML297 (33ug) produced a robust analgesic effect (+5 sec above baseline) compared to vehicle treated animals as well as prevented PGE2‐evoked thermal allodynia. To further corroborate the importance of GIRK channels in peripheral KOR mediated antinociception, we tested effects of siRNA‐mediated knockdown of GIRK2 channel subunits following local administration of GIRK2 Accell‐siRNA (Dharmacon) into the hindpaw. After two daily i.pl. injections of GIRK2 siRNA, neither baseline PWL nor PGE2‐evoked thermal allodynia were altered, however, U50488‐mediated reduction in PGE2‐evoked thermal allodynia was lost. We verified knockdown of the GIRK2 subunit in hindpaws by RT‐qPCR and immunohistochemistry. Compared to the contralateral (CONTRA) hindpaw, GIRK2 mRNA was decreased significantly in the ipsilateral (IPSI) hindpaw. Further, there was no difference between IPSI and CONTRA hindpaws in the immunofluorescence for PGP9.5, a marker used to identify neuronal processes, or DAPI, a cell nuclei marker. By contrast, GIRK2 immunofluorescence was significantly decreased in IPSI hindpaws compared to CONTRA hindpaws suggesting that the expression of GIRK2 was indeed reduced. Together these results suggest that peripherally mediated antinociception in response to activation of KOR is due, in large part, to activation of GIRK channels.Support or Funding InformationThis work is supported by NIH/NIDA RO1 DA 038645 and NIH/NIGMS RO1 GM 106035.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.